Compositions and methods of identifying tumor specific neoantigens

ABSTRACT

The present invention related to immunotherapeutic peptides and their use in immunotherapy, in particular the immunotherapy of cancer. Specifically, the invention provides a method of identifying tumor specific neoantigens that alone or in combination with other tumor-associated peptides serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor responses.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/108,610 filed May 16, 2011, and which claims the benefits of U.S.provisional application No. 61/334,866, filed May 14, 2010, which isincorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file name “39564-502001US_ST25.txt”, which wascreated on Jul. 19, 2011 and is 73 KB in size, are hereby incorporatedby reference it their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the identification of tumorspecific neoantigens and the uses of these neoantigens to produce cancervaccines.

BACKGROUND OF THE INVENTION

Tumor vaccines are typically composed of tumor antigens andimmunostimulatory molecules (e.g. cytokines or TLR ligands) that worktogether to induce antigen-specific cytotoxic T cells (CTLs) thatrecognize and lyse tumor cells. At this time, almost all vaccinescontain either shared tumor antigens or whole tumor cell preparations(Gilboa, 1999). The shared tumor antigens are immunogenic proteins withselective expression in tumors across many individuals and are commonlydelivered to patients as synthetic peptides or recombinant proteins(Boon et al., 2006). In contrast, whole tumor cell preparations aredelivered to patients as autologous irradiated cells, cell lysates, cellfusions, heat-shock protein preparations or total mRNA (Parmiani et al.,2007). Since whole tumor cells are isolated from the autologous patient,the cells express patient-specific tumor antigens as well as sharedtumor antigens. Finally, there is a third class of tumor antigens thathas rarely been used in vaccines due to technical difficulties inidentifying them (Sensi et al. 2006). This class consists of proteinswith tumor-specific mutations that result in altered amino acidsequences. Such mutated proteins have the potential to: (a) uniquelymark a tumor (relative to non-tumor cells) for recognition anddestruction by the immune system (Lennerz et al., 2005); (b) avoidcentral and sometimes peripheral T cell tolerance, and thus berecognized by more effective, high avidity T cells receptors (Gotter etal., 2004).

Thus a need exists for a method of identifying neoepitopes that areuseful as tumor vaccines.

SUMMARY OF THE INVENTION

The present invention relates in part to the discovery of a method ofidentifying peptides that are capable of celicting a tumor specificT-cell response.

In one aspect the invention provides methods of identifying a neoantigenby identifying a tumor specific mutation in an expressed gene of asubject having cancer. In some aspects when the mutation is a pointmutation the method further comprises identifying the mutant peptidehaving the mutation. Preferably the mutant peptide binds to a class IHLA protein with a greater affinity than a wild-type peptide and has anIC50 less than 500 nm; In other aspects when the mutation is asplice-site, frameshift, read-through or gene-fusion mutation the methodfurther comprise identifying the mutant polypeptide encoded by themutation. Preferably, the mutant polypeptide binds to a class I HLAprotein.

Optionally, the method further includes selecting peptides orpolypeptides that activate anti-tumor CD8 T cells.

The mutant peptide or polypeptide preferably binds to a class I HLAprotein with a greater affinity than a wild-type peptide and has an IC50less than 500 nM. Preferably, the peptide or polypeptide has an IC50less than 250 nM. More preferably, the peptide or polypeptide has anIC50 less than 100 nM. Most preferably, the peptide or polypeptide hasan IC50 less than 50 nM.

The mutant peptide is about 8-10 amino acids in length. In anotheraspect is about 8-50 amino acids in length. For example, mutant peptideis greater than 10 amino acids in leghth, greater than 15 amino acids inlength, greater than 20 amino acids in length, greater than 30 aminoacids in length. Preferably the mutant peptides is about 24-40 aminoacids in length.

In a further aspect the invention provides methods of inducing a tumorspecific immune response in a subject by administering one or morepeptides or polypeptides identified according to the methods of theinvention and an adjuvant. The adjuvant is for example, a TLR-basedadjuvant or a mineral oil based adjuvant. In some aspects the peptide orpolypeptide and TLR-based adjuvant is emulsified with a mineral oilbased adjuvant. Optionally, the method further includes administering ananti-immunosuppressive agent such as an anti-CTLA-4 antibody, an anti-PD1 antibody an anti-PD-L1 antibody an anti-CD25 antibody or an inhibitorof IDO.

In yet another aspect the invention provides methods of inducing a tumorspecific immune response in a subject by administering to the subjectautologous dendritic cells or antigen presenting cells that have beenpulsed with one or more of the peptides or polypeptides identifiedaccording to the methods of the inventions. Optionally, the methodfurther includes administering an adjuvant such as for example, aTLR-based adjuvant or a mineral oil based adjuvant. In some aspects thepeptide or polypeptide and TLR-based adjuvant is emulsified with amineral oil based adjuvant. In some embodiments the method furtherincludes administering an anti-immunosuppressive agent.Anti-immunosuppressive agents include for example an anti-CTLA-4antibody, an anti-PD 1 antibody an anti-PD-L1 antibody an anti-CD25antibody or an inhibitor of IDO.

In another aspect the invention provides a method of vaccinating ortreating a subject for cancer by identifying a plurality of tumorspecific mutations in an expressed gene of the subject, identifyingmutant peptides or polypeptides having the identified tumor specificmutations, selecting one or more of the identified mutant peptide orpolypeptides that binds to a class I HLA protein preferably with agreater affinity than a wild-type peptide and is capable of activatinganti-tumor CD8 T-cells, and administering to the subject the one or moreselected peptides, polypeptides or autologous dendritic cells or antigenpresenting cells pulsed with the one or more identified peptides orpolypeptides. The mutant peptide is about 8-10 amino acids in length. Inanother aspect is about 8-50 amino acids in length. For example, mutantpeptide is greater than 10 amino acids in length, greater than 15 aminoacids in length, greater than 20 amino acids in length, greater than 30amino acids in length. Preferably, the mutant peptides is about 24-40amino acids in length.

Optionally, the method further includes administering an adjuvant suchas for example, a TLR-based adjuvant or a mineral oil based adjuvant. Insome aspects the peptide or polypeptide and TLR-based adjuvant isemulsified with a mineral oil based adjuvant. In some embodiments themethod further includes administering an anti-immunosuppressive agent.Anti-immunosuppressive agents include for example an anti-CTLA-4antibody, an anti-PD 1 antibody an anti-PD-L1 antibody an anti-CD25antibody or an inhibitor of IDO.

The method of claim 22, wherein said subject has received ahematopoietic stem cell transplant.

The subject is a human, dog, cat, or horse. The cancer is breast cancer,ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastriccancer, colon cancer, testicular cancer, head and neck cancer,pancreatic cancer, brain cancer, melanoma lymphoma, such as B-celllumphoma or leukemia, such as cute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, or T celllymphocytic leukemia.

Also included in the invention are pharmaceutical compositionscontaining the peptide or polypeptide identified according the methodsof the invention and a pharmaceutically acceptable carrier.

For example, the invention provides a composition containing least twodistinct SF3B1 peptides wherein each peptide is equal to or less than 50amino acids in length and contains

-   -   a leucine at amino acid position 625;    -   a histidine at amino acid position 626;    -   a glutamic acid at amino acid position 700;    -   an aspartic acid at amino acid position 742; or    -   an arginine at amino acid position 903, when numbered in        accordance with wild-type SF3B1.

The invention also provides a composition containing at least twodistinct MYD88 peptides where each peptide is equal to or less than 50amino acids in length and contains a threonine at amino acid position232; a leucine at amino acid position 258; or

a proline at amino acid position 265, when numbered in accordance withwild-type MYD88

The invention further provides composition containing at least twodistinct TP53 peptides where each peptide is equal to or less than 50amino acids in length and contains an arginine at amino acid position111; an arginine at amino acid position 215; a serine at amino acidposition 238; a glutamine at amino acid position 248; a phenylalanine atamino acid position 255; a cysteine at amino acid position 273 or anasparagine at amino acid position 281, when numbered in accordance withwild-type TP53.

The invention further provides composition containing at least twodistinct ATM peptides wherein each peptide is equal to or less than 50amino acids in length and contain a phenylalanine at amino acid position1252; an arginine at amino acid position 2038; a histidine at amino acidposition 2522; or a cysteine at amino acid position 2954, when numberedin accordance with wild-type ATM.

A composition comprising at least two distinct Abl peptides wherein eachpeptide is equal to or less than 50 amino acids in length and contains avaline at amino acid position 244;

a valine at amino acid position 248;a glutamic acid at amino acid position 250; an alanine at amino acidposition 250; a histidine at amino acid position 252; an arginine atamino acid position 252; a phenylalanine at amino acid position 253; ahistidine at amino acid position 253; a lysine at amino acid position255; a valine at amino acid position 255; a glycine at amino acidposition 276; an isoleucine at amino acid position 315; an asparagine atamino acid position 315; a leucine at amino acid position 317; athreonine at amino acid position 343; a threonine at amino acid position351; a glycine at amino acid position 355; a valine at amino acidposition 359; an alanine at amino acid position 359; an isoleucine atamino acid position 379; a leucine at amino acid position 382; amethionine at amino acid position 387; a proline at amino acid position396; an arginine at amino acid position 396; a tyrosine at amino acidposition 417; or a serine at amino acid position 486, when numbered inaccordance with wild-type ABL.

Further included in the invention is a composition containing at leasttwo distinct FBXW7 peptides where each peptide is equal to or less than50 amino acids in length and contains a leucine at amino acid position280; a histidine at amino acid position 465; a cysteine at amino acidposition 505; or a glutamic acid at amino acid position 597, whennumbered in accordance with wild-type FBXW7.

In a further a aspect the invention provides a composition containing atleast two distinct MAPK1 peptides where each peptide is equal to or lessthan 50 amino acids in length and contains an asparagine at amino acidposition 162; a glycine at amino acid position 291; or a phenylalanineat amino acid position 316, when numbered in accordance with wild-typeMAPK1.

The invention also provides a composition containing at least twodistinct GNB1 peptides wherein each peptide is equal to or less than 50amino acids in length and contains a threonine at amino acid position180, when numbered in accordance with wild-type GNB1.

Also provided by the invention is a method of treating a subject with animatinib resistant tumor to a HLA-A3 positive subject a composition ofBcr-abl peptide equal to or less than 50 amino acid in length thatcontains a lysine at position 255 when numbered in accordance withwild-type bcr-abl.

Further provided by the invention, is method of treating a subject withan imatinib resistant tumor comprising administering to the ubject oneor more peptides containing a bcr-abl mutation where the peptide isequal to or less than 50 amino acid and binds to a class I HLA proteinwith an IC50 less than 500 nm.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the balance of specificity and autoimmune toxicity using 3classes of antigens for tumor vaccines. Whole tumor cells may be theleast specific antigen formulation for tumor vaccines since the full setof protein antigens expressed in tumor cells include thousands ofproteins that are also present in other cells of the body. Overexpressedtumor antigens are slightly more specific because they have beenselected for much higher and more selective expression in tumorscompared to other cells in the body. Nevertheless, it is impossible totest every cell in the body for the expression of these antigens andthere is a substantial risk that other cells express them. Finally,mutated proteins generate neoepitopes that are present only in tumorcells and provide the greatest level of specificity.

FIG. 2 is a schema for a personalized neoantigen vaccination strategythat can be applied to the treatment of any cancer. We also highlightthe possibility of applying this strategy in two unique scenarios. Inthe first case, a patient is vaccinated in the early period followinghematopoietic stem cell transplantation (HSCT) (e.g. as is done for CLL,CML and other leukemias). The early post-HSCT period is a uniquetherapeutic setting as the immune system is competent due toreconstitution with HSCT, thus overcoming tumor- or treatment-inducedhost immune defects. Moreover, the abundance of homeostatic cytokines ina lymphopenia milieu, such as in the early post-HSCT setting, cancontribute to rapid expansion of T cells. In the second case, a patientis vaccinated early in the disease course when immune competence may bemore intact in the early stages of disease, before impairment byexposure to chemotherapy (e.g. for solid or hematopoeitic tumors). Sincethe immune system is likely to be most active in these two specificsituations, we suggest that these are the ideal situations for applyingtumor vaccination strategies.

FIG. 3 shows a strategy for identifying tumor neoepitopes is describedin 3 steps: (1) using sequencing technologies, detect gene mutationsthat are present in tumor but not germline DNA of a single patient; (2)using prediction algorithms, predict whether mutated peptides have thepotential to bind personal HLA allele; these predicted peptides mayoptionally be tested experimentally for binding to appropriate HLAproteins. In addition, these genes must also be expressed in tumorcells. (3) generate T cells ex vivo and test whether they are able torecognize cells expressing the mutated protein; alternatively, massspectrometry can be used to detect peptides eluted from tumor cellsurface HLA proteins. For chronic lymphocytic leukemia, our studies todate demonstrate that there are an average of 23 protein-alteringmutations per patient, 46 predicted binding mutant peptides and 15-25validated binding mutant peptides. Of these, we anticipate that ˜7-12peptides are expressed and processed in tumor cells (though this maydiffer across tumors and patients).

FIG. 4 shows five classes of mutations generate potential tumorneoepitopes. New tumor-specific epitopes can arise as a result ofmissense, splice-site, frameshift or read-through point mutations (redasterisk), or from the fusion of two genes (or within the same gene). Inparticular, splice-site, frameshift, read-through mutations and genefusions can each generate novel stretches of amino acids (in magenta)that are normally not translated, but now are expressed and translatedas a result of mutation. Missense mutations lead to peptides with singleamino acid changes.

FIG. 5 shows the frequency of mutations per class in CLL patients. Ourstudies applying next-generation sequencing to a series of 7 CLL tumorsreveal that CLL cells harbor many mutations that provide a rich sourceof possible mutated peptides. We observe that the total number ofnonsilent gene alterations in CLL ranged from 17-155 per individual, themajority of which were somatically altered point mutations (missense).The tumors of 4 patients also harbored splice-site mutations; for 3patients, novel gene fusions were identified by RNA sequencing.

FIG. 6 shows data from automated predictions (Step 2A of the strategy inFIG. 3) of peptide binding (for peptides that harbor a specific missensemutation) against each of a patient's 6 HLA (MHC Class I) alleles.Magenta=strong binders; green=intermediate binders.

FIG. 7 shows methods for conifrming RNA expression of mutated genes(Step 2B of the strategy in FIG. 3). A. For CLL patient 7, we found thatmore than half of the mutated genes with predicted HLA-binding peptideswere expressed at the RNA level. B. We have also used RNA pyrosequencingto detect expressed RNAs harboring specific mutations found in DNA. C.We can validate novel gene fusions that were seen by DNA sequencingusing PCR-TOPO cloning of the breakpoint region (depicted is a fusiondiscovered for patient 2).

FIG. 8A-C shows a method and data for experimental validation ofHLA-peptide binding (Step 2C of the strategy in FIG. 3). A. Schema forexperimental validation of peptide binding to specific HLA alleles. B.Summary of candidate mutated peptides identified in patients 1 and 2.Shaded cells indicate that analysis is in progress. C. Data forpredicted vs experimentally verified binding affinity of peptidesgenerated from gene alterations (missense mutation or gene fusion) forpatient 2. A prediction cutoff of IC₅₀<120 nM (solid vertical line onleft) results in all peptides showing experimental binding to class IHLA.

FIG. 9 shows predicted differential binding of mutated vs germline (i.ealso called parental, wild type or normal) peptides to HLA alleles. 12of 25 predicted HLA binding mutated peptides of Pt 2 have >2 foldgreater binding (cutoff=red dotted line) than parental peptides. Thisfurther increases the specificity of mutated peptides. Mutated peptidesare specific for two reasons: first, many of the T cell receptors thatrecognize a mutated peptide are not likely to detect the wild typeparental peptide; second, some of the mutated peptides can bind HLA withhigher affinity than the parental peptide. Since the first propertycannot be computationally predicted, we will focus on predicting thesecond property and selecting for inclusion in vaccines only thosepeptides that show higher binding to HLA for mutated relative to wildtype peptides.

FIG. 10 shows T cell reactivity against a candidate personal CLLneoepitope (Step 3 of the strategy in FIG. 3). We observed that T cellsisolated from patient 1 post-therapy can detect a specific mutated TLK2peptide (peptide #7) (using the Elispot assay).

FIG. 11 shows that BCR-ABL mutations generate many peptides predicted tobind HLA-A and HLA-B alleles. By applying the NetMHC predictionalgorithm (Nielsen et al. PLoS One. 2007, 2(8):e796), we predictedpeptides generated from the BCR-ABL mutations with potential to bind to8 common HLA-A and -B alleles. The most common BCR-ABL mutations areordered in decreasing frequency (from left to right), and predicted IC50of various class I MHC binding peptides are depicted. In total, wepredicted 84 peptides to bind with good affinity, defined as an IC50 ofless than 1000, across a wide range of HLA alleles. Of all the predictedpeptides, 24 of 84 (29%) were predicted to be strong binders with anIC50<50. 42 peptides (50%) were intermediate binders, defined as IC50between 50 and 500. 18 peptides (21%) were weak binders defined as IC50between 500 and 1000.

FIG. 12A-D shows BCR-ABL peptide harboring the E255K mutation binds HLAproteins and is associated with specific, polyfunctional T cells presentin CML patients. A. Experimentally-derived binding scores of E255K-B(and parental peptide) to HLA A3 and supertype members. B. In CD8+ Tcells expanded from a HLAA3+E255K+patient following HSCT, we detectedIFNgamma secretion against the E255K-B (MUT) peptide and A3+ expressingAPCs expressing the E255K minigene (MG). This response was abrogated inthe presence of the class I blocking antibody (w6/32). C.IFNgamma-secreting cells were also tetramer+ for the mutated peptide andwere (D) polyfunctional, secreting IP 10, TNFalpha and GM-CSF (based onthe Luminex assay).

FIG. 13A-C shows that patient-derived T cell clones can recognizetumor-specific epitopes and kill cells presenting these epitopes. A.Reactivity to the CD8+ T cell epitope of CML66 (peptide 66-72C) isrestricted by HLA B-4403. B. CML66 mRNA can be efficiently nucleofectedinto CD40L-expanded B cells. C. CML66-specific CD8+ T cells arecytotoxic to CD40L B cells expressing CML66 by RNA nucleofection or bypeptide pulse, but not control targets.

FIG. 14A-C shows significantly mutated genes in CLL. A. The 9 mostsignificantly mutated genes among 64 CLL samples. N—total coveredterritory in base pairs across 64 sequenced samples. p- and q-valueswere calculated by comparing the probability of seeing the observedconstellation of mutations to the background mutation rates calculatedacross the dataset. Red bars—genes not previously known to be mutated inCLL; grey bars—genes in which mutation in CLL has been previouslyreported. B-C. Type (missense, splice-site, nonsense) and location ofmutations in ATM, SF3B1, TP53, MYD88, FBXW7, DDX3X, MAPK1, and GNB1discovered among the 64 CLLs (position and mutation in CLL samples shownabove the gene) compared to previously reported mutations in literatureor in the COSMIC database (lines show position of mutations below thegene).

FIG. 15 shows that SF3B1 is expressed in CLL samples (7th column ingraph) and has higher expression than many control cells, including:PBMC, M: monocyte, CC: cancer cell lines (includes K562, Jurkat. IM9,MCF-7, Hela, Ovcar, RPMI, OTM, MCF-CAR, KM12BM and MM1S).

FIG. 16 shows that SF3B1 mutations generate peptides that are predictedto bind to patient-specific HLA alleles. For example, one peptide thatincludes the common SF3B1 K700E mutation is predicted to bind HLAstrongly.

DETAILED DESCRIPTION OF THE INVENTION

One of the critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly restrictedtumor antigens to avoid autoimmunity. Tumor neoantigens, which arise asa result of genetic change within malignant cells, represent the mosttumor-specific class of antigens. Neoantigens have rarely been used invaccines due to technical difficulties in identifying them. Our approachto identify tumor-specific neoepitopes involves three steps. (1)identification of DNA mutations using whole genome or whole exome (i.e.only captured exons) or RNA sequencing of tumor versus matched germlinesamples from each patient; (2) application of validated peptide-MHCbinding prediction algorithms to generate a set of candidate T cellepitopes that may bind patient HLA alleles and are based on non-silentmutations present in tumors; and (3) optional demonstration ofantigen-specific T cells against mutated peptides or demonstration thata candidate peptide is bound to HLA proteins on the tumor surface.

Accordingly, the present invention relates to methods for identifyingand/or detecting T-cell epitopes of an antigen. Specifically, theinvention provides method of identifying and/or detecting tumor specificneoantigens that are useful in inducing a tumor specific immune responsein a subject.

In particular, the invention provides a method of vaccinating ortreating a subject by identifying a plurality of tumor specificmutations in the genome of a subject. Mutant peptides and polypeptideshaving the identified mutations and that binds to a class I HLA proteinare selected. Optionally, these peptide and polypeptides binds to aclass I HLA proteins with a greater affinity than the wild-type peptideand/or are capable of activating anti-tumor CD8 T-cells These peptidesare admistered to the subject. Alternatively, autologousantigen-presenting cells that have been pulsed with the peptides areadministered.

The importance of mutated antigens, or neoepitopes, in the immunecontrol of tumors has been appreciated in seminal studies showing that:(a) mice and humans often mount T cell responses to mutated antigens(Parmiani et al., 2007; Sensi and Anichini, 2006); (b) mice can beprotected from a tumor by immunization with a single mutated peptidethat is present in the tumor (Mandelboim et al., 1995); (c) spontaneousor vaccine-mediated long-term melanoma survivors mount strong memorycytotoxic T cell (CTL) responses to mutated antigens (Huang et al.,2004; Lennerz et al., 2005; Zhou et al., 2005a); (d) finally, follicularlymphoma patients show molecular remission when immunized withpatient-specific mutated immunoglobulin proteins that are present inautologous tumor cells. (Baskar et al., 2004). Furthermore, the CTLresponses in these patients are directed toward the mutated rather thanshared regions of the immunoglobulin protein. Additionally, such mutatedpeptides have the potential to: (a) uniquely mark a tumor forrecognition and destruction by the immune system, thus reducing the riskfor autoimmunity; and (b) avoid central and peripheral T cell tolerance,allowing the antigen to be recognized by more effective, high avidity Tcells receptors. (FIG. 1)

Identical mutations in any particular gene are rarely found acrosstumors (and are even at low frequency for the most common drivermutations). Thus, the methods of the present invention willcomprehensively identify patient-specific tumor mutations. Using highlyparallel sequencing technologies, HLA-peptide binding prediction toolsand biochemical assays the methods of the invention will allow: (1)comprehensive identification of mutated peptides that are expressed andbind HLA proteins present in a patient's tumor; (2) monitoring of thenatural immune response of cancer patients to these identifiedneoepitopes; (3) determining whether cytotoxic T cells that recognizethese peptides in the context of patient HLA proteins can selectivelylyse autologous tumor cells ex vivo. This strategy addresses severalfundamental questions related to how the immune system of cancerpatients interacts with tumor neoepitopes. These include: which and whatfraction of tumor neoepitopes are detected by T cells, how many T cellprecursors are able to respond to neoepitopes, how frequent areneoepitope-specific memory and effector T cells in circulation and inthe tumor, how much avidity do T cells have for these epitopes, areneoepitope-specific T cells functional? The answers to these questionsprovide both the justification and strategy for using tumor neoepitopesin human vaccines.

The immune system of a human can be classified into two functionalsubsystems, i.e., the innate and the acquired immune system. The innateimmune system is the first line of defense against infections, and mostpotential pathogens are rapidly neutralized before they can cause, forexample, a noticeable infection. The acquired immune system reacts tomolecular structures, referred to as antigens, of the intrudingorganism. There are two types of acquired immune reactions, i.e. thehumoral immune reaction and the cell-mediated immune reaction. In thehumoral immune reaction, the antibodies secreted by B cells into bodilyfluids bind to pathogen-derived antigens, leading to the elimination ofthe pathogen through a variety of mechanisms, e.g. complement-mediatedlysis. In the cell-mediated immune reaction, T-cells capable ofdestroying other cells are activated. If, for example, proteinsassociated with a disease are present in a cell, they are, within thecell, fragmented proteolytically to peptides. Specific cell proteinsthen attach themselves to the antigen or peptide formed in this mannerand transport them to the surface of the cell, where they are presentedto the molecular defense mechanisms, in particular T-cells, of the body.Cytotoxic T cells recognize these antigens and kill the cells thatharbor the antigens.

The molecules which transport and present peptides on the cell surfaceare referred to as proteins of the major histocompatibility complex(MHC). The MHC proteins are classified into MHC proteins of class I andof class II. The structures of the proteins of the two MHC classes arevery similar; however, they differ quite considerably in their function.Proteins of MHC class I are present on the surface of almost all cellsof the body, including most tumor cells. The proteins of MHC class I areloaded with antigens that usually originate from endogenous proteins orfrom pathogens present inside cells, and are then presented to cytotoxicT-lymphocytes (CTLs). The MHC proteins of class II are only present ondendritic cells, B-lymphocytes, macrophages and other antigen-presentingcells. They present mainly peptides, which are processed from externalantigen sources, i.e. outside of the cells, to T-helper (Th) cells. Mostof the peptides bound by the MHC proteins of class I originate fromcytoplasmic proteins produced in the healthy host organism itself anddon't normally stimulate an immune reaction. Accordingly, cytotoxicT-lymphocytes which recognize such self-peptide-presenting MHC moleculesof class I are deleted in the thymus or, after their release from thethymus, are deleted or inactivated, i.e. tolerized. MHC molecules areonly capable of stimulating an immune reaction when they presentpeptides to non-tolerized cytotoxic T-lymphocytes. CytotoxicT-lymphocytes have, on their surface, both T-cell receptors (TCR) andCD8 molecules. T-Cell receptors are capable of recognizing and bindingpeptides complexed with the molecules of MHC class I. Each cytotoxicT-lymphocyte expresses a unique T-cell receptor which is capable ofbinding specific MHC/peptide complexes.

The peptides attach themselves to the molecules of MHC class I bycompetitive affinity binding within the endoplasmic reticulum, beforethey are presented on the cell surface. Here, the affinity of anindividual peptide is directly linked to its amino acid sequence and thepresence of specific binding motifs in defined positions within theamino acid sequence. If the sequence of such a peptide is known, it ispossible, for example, to manipulate the immune system against diseasedcells using, for example, peptide vaccines.

Using computer algorithms, it is possible to predict potential T-cellepitopes, i.e. peptide sequences, which are bound by the MHC moleculesof class I or class II in the form of a peptide-presenting complex andthen, in this form, recognized by the T-cell receptors of T-lymphocytes.Currently, use is made, in particular, of two programs, namely SYFPEITHI(Rammensee et al., Immunogenetics, 50 (1999), 213-219) and HLA_BIND(Parker et al., J. Immunol., 152 (1994), 163-175). The peptide sequencesdetermined in this manner, which potentially may bind to MHC moleculesof class I, then have to be examined in vitro for their actual bindingcapacity.

The technical object of the present invention is to provide an improvedmethod for identifying and screening potential T-cell epitopes presentin tumor cells, which method allows for simultaneous and rapidexamination of a large number of peptide sequences, for their capabilityof binding to specific MHC molecules.

In the present invention, the technical object on which it is based isachieved by providing a method for identifying and/or detecting mutatedantigens that are present in tumors but not in normal tissue. The methoduses massively parallel genomic sequencing of the entire coding portionof a cancer patient genome to identify the specific mutated genes in atumor. In order to narrow down the mutant peptides to those withpotential to bind more strongly to HLA than the wild type peptides andthus confer tumor specificity, well-established algorithms will be usedto predict peptides that bind any of the 6 unique class I HLA alleles ofeach patient and a predicted IC50 for all 9- or 10-mer peptides withtumor-specific mutant residues vs. those with the germline residue willbe calculated. Typically, peptides with predicted IC50<50 nM, aregenerally considered medium to high affinity binding peptides and willbe selected for testing their affinity empirically using biochemicalassays of HLA-binding. Finally, it will be determined whether the humanimmune system can mount effective immune responses against these mutatedtumor antigens and thus effectively kill tumor but not normal cells.

DEFINITIONS

A “T-cell epitope” is to be understood as meaning a peptide sequencewhich can be bound by the MHC molecules of class I or II in the form ofa peptide-presenting MHC molecule or MHC complex and then, in this form,be recognized and bound by cytotoxic T-lymphocytes or T-helper cells,respectively

A “receptor” is to be understood as meaning a biological molecule or amolecule grouping capable of binding a ligand. A receptor may serve, totransmit information in a cell, a cell formation or an organism. Thereceptor comprises at least one receptor unit and preferably tworeceptor units, where each receptor unit may consist of a proteinmolecule, in particular a glycoprotein molecule. The receptor has astructure which complements that of a ligand and may complex the ligandas a binding partner. The information is transmitted in particular byconformational changes of the receptor following complexation of theligand on the surface of a cell. According to the invention, a receptoris to be understood as meaning in particular proteins of MHC classes Iand II capable of forming a receptor/ligand complex with a ligand, inparticular a peptide or peptide fragment of suitable length.

A “ligand” is to be understood as meaning a molecule which has astructure complementary to that of a receptor and is capable of forminga complex with this receptor. According to the invention, a ligand is tobe understood as meaning in particular a peptide or peptide fragmentwhich has a suitable length and suitable binding motives in its aminoacid sequence, so that the peptide or peptide fragment is capable offorming a complex with proteins of MHC class I or MHC class II.

A “receptor/ligand complex” is also to be understood as meaning a“receptor/peptide complex” or “receptor/peptide fragment complex”, inparticular a peptide- or peptide fragment-presenting MHC molecule ofclass I or of class II.

“Proteins or molecules of the major histocompatibility complex (MHC)”.“MHC molecules”, “MHC proteins” or “HLA proteins” are to be understoodas meaning, in particular, proteins capable of binding peptidesresulting from the proteolytic cleavage of protein antigens andrepresenting potential T-cell epitopes, transporting them to the cellsurface and presenting them there to specific cells, in particularcytotoxic T-lymphocytes or T-helper cells. The major histocompatibilitycomplex in the genome comprises the genetic region whose gene productsexpressed on the cell surface are important for binding and presentingendogenous and/or foreign antigens and thus for regulating immunologicalprocesses. The major histocompatibility complex is classified into twogene groups coding for different proteins, namely molecules of MHC classI and molecules of MHC class II. The molecules of the two MHC classesare specialized for different antigen sources. The molecules of MHCclass I present endogenously synthesized antigens, for example viralproteins and tumor antigens. The molecules of MHC class II presentprotein antigens originating from exogenous sources, for examplebacterial products. The cellular biology and the expression patterns ofthe two MHC classes are adapted to these different roles.

MHC molecules of class I consist of a heavy chain and a light chain andare capable of binding a peptide of about 8 to 11 amino acids, butusually 9 or 10 amino acids, if this peptide has suitable bindingmotifs, and presenting it to cytotoxic T-lymphocytes. The peptide boundby the MHC molecules of class I originates from an endogenous proteinantigen. The heavy chain of the MHC molecules of class I is preferablyan HLA-A, HLA-B or HLA-C monomer, and the light chain isβ-2-microglobulin.

MHC molecules of class II consist of an α-chain and a β-chain and arecapable of binding a peptide of about 15 to 24 amino acids if thispeptide has suitable binding motifs, and presenting it to T-helpercells. The peptide bound by the MHC molecules of class II usuallyoriginates from an extracellular of exogenous protein antigen. Theα-chain and the β-chain are in particular HLA-DR, HLA-DQ and HLA-DPmonomers.

A “vaccine” is to be understood as meaning a composition for generatingimmunity for the prophylaxis and/or treatment of diseases. Accordingly,vaccines are medicaments which comprise antigens and are intended to beused in humans or animals for generating specific defense and protectivesubstance by vaccination.

“Isolated” means that the polynucleotide or polypeptide or fragment,variant, or derivative thereof has been essentially removed from otherbiological materials with which it is naturally associated, oressentially free from other biological materials derived, e.g., from arecombinant host cell that has been genetically engineered to expressthe polypeptide of the invention.

“Neoantigen” means a class of tumor antigens which arises fromtumor-specific mutations in expressed protein.

“Purified” means that the polynucleotide or polypeptide or fragment,variant, or derivative thereof is substantially free of other biologicalmaterial with which it is naturally associated, or free from otherbiological materials derived, e.g., from a recombinant host cell thathas been genetically engineered to express the polypeptide of theinvention. That is, e.g., a purified polypeptide of the presentinvention is a polypeptide that is at least about 70-100% pure, i.e.,the polypeptide is present in a composition wherein the polypeptideconstitutes about 70-100% by weight of the total composition. In someembodiments, the purified polypeptide of the present invention is about75%-99% by weight pure, about 80%-99% by weight pure, about 90-99% byweight pure, or about 95% to 99% by weight pure.

Identification of Tumor Specific Mutations

The present invention is based, on the identification of certainmutations (e.g., the variants or alleles that are present in cancercells). In particular, these mutations are present in the genome ofcancer cells of a subject having cancer but not in normal tissue fromthe subject.

Genetic mutations in tumors would be considered useful for theimmunological targeting of tumors if they lead to changes in the aminoacid sequence of a protein exclusively in the tumor. Useful mutationsinclude: (1) non-synonymous mutations leading to different amino acidsin the protein; (2) read-through mutations in which a stop codon ismodified or deleted, leading to translation of a longer protein with anovel tumor-specific sequence at the C-terminus; (3) splice sitemutations that lead to the inclusion of an intron in the mature mRNA andthus a unique tumor-specific protein sequence; (4) chromosomalrearrangements that give rise to a chimeric protein with tumor-specificsequences at the junction of 2 proteins (i.e., gene fusion); (5)frameshift mutations or deletions that lead to a new open reading framewith a novel tumor-specific protein sequence.

Peptides with mutations or mutated polypeptides arising from forexample, splice-site, frameshift, readthrough, or gene fusion mutationsin tumor cells may be identified by sequencing DNA, RNA or protein intumor versus normal cells.

Also within the scope of the inventions are peptides including previousidentified tumor specific mutations. Know tumor mutation can be found atthe Catalogue of Somatic Mutations in Cancer (COSMIC).

A variety of methods are available for detecting the presence of aparticular mutation or allele in an individual's DNA or RNA.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms in genomic DNA or celluar RNA. In oneembodiment, the single base polymorphism can be detected by using aspecialized exonuclease-resistant nucleotide, as disclosed, e.g., inMundy, C. R. (U.S. Pat. No. 4,656,127). According to the method, aprimer complementary to the allelic sequence immediately 3′ to thepolymorphic site is permitted to hybridize to a target molecule obtainedfrom a particular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA® isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA® in that they allrely on the incorporation of labeled deoxynucleotides to discriminatebetween bases at a polymorphic site. In such a format, since the signalis proportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

A number of initiatives are currently underway to obtain sequenceinformation directly from millions of individual molecules of DNA or RNAin parallel. Real-time single molecule sequencing-by-synthesistechnologies rely on the detection of fluorescent nucleotides as theyare incorporated into a nascent strand of DNA that is complementary tothe template being sequenced. In one method, oligonucleotides 30-50bases in length are covalently anchored at the 5′ end to glass coverslips. These anchored strands perform two functions. First, they act ascapture sites for the target template strands if the templates areconfigured with capture tails complementary to the surface-boundoligonucleotides. They also act as primers for the template directedprimer extension that forms the basis of the sequence reading. Thecapture primers function as a fixed position site for sequencedetermination using multiple cycles of synthesis, detection, andchemical cleavage of the dye-linker to remove the dye. Each cycleconsists of adding the polymerase/labeled nucleotide mixture, rinsing,imaging and cleavage of dye. In an alternative method, polymerase ismodified with a fluorescent donor molecule and immobilized on a glassslide, while each nucleotide is color-coded with an acceptor fluorescentmoiety attached to a gamma-phosphate. The system detects the interactionbetween a fluorescently-tagged polymerase and a fluorescently modifiednucleotide as the nucleotide becomes incorporated into the de novochain. Other sequencing-by-synthesis technologies also exist.

Preferably, any suitable sequencing-by-synthesis platform can be used toidentify mutations. As described above, four majorsequencing-by-synthesis platforms are currently available: the GenomeSequencers from Roche/454 Life Sciences, the 1 G Analyzer fromIllumina/Solexa, the SOLiD system from Applied BioSystems, and theHeliscope system from Helicos Biosciences. Sequencing-by-synthesisplatforms have also been described by Pacific BioSciences and VisiGenBiotechnologies. Each of these platforms can be used in the methods ofthe invention. In some embodiments, a plurality of nucleic acidmolecules being sequenced is bound to a support (e.g., solid support).To immobilize the nucleic acid on a support, a capturesequence/universal priming site can be added at the 3′ and/or 5′ end ofthe template. The nucleic acids may be bound to the support byhybridizing the capture sequence to a complementary sequence covalentlyattached to the support. The capture sequence (also referred to as auniversal capture sequence) is a nucleic acid sequence complementary toa sequence attached to a support that may dually serve as a universalprimer.

As an alternative to a capture sequence, a member of a coupling pair(such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotinpair as described in, e.g., US Patent Application No. 2006/0252077) maybe linked to each fragment to be captured on a surface coated with arespective second member of that coupling pair.

Subsequent to the capture, the sequence may be analyzed, for example, bysingle molecule detection/sequencing, e.g., as described in the Examplesand in U.S. Pat. No. 7,283,337, including template-dependentsequencing-by-synthesis. In sequencing-by-synthesis, the surface-boundmolecule is exposed to a plurality of labeled nucleotide triphosphatesin the presence of polymerase. The sequence of the template isdetermined by the order of labeled nucleotides incorporated into the 3′end of the growing chain. This can be done in real time or can be donein a step-and-repeat mode. For real-time analysis, different opticallabels to each nucleotide may be incorporated and multiple lasers may beutilized for stimulation of incorporated nucleotides.

Any cell type or tissue may be utilized to obtain nucleic acid samplesfor use in the diagnostics described herein. In a preferred embodiment,the DNA or RNA sample is obtained from a tumor or a bodily fluid, e.g.,blood, obtained by known techniques (e.g. venipuncture) or saliva.Alternatively, nucleic acid tests can be performed on dry samples (e.g.hair or skin).

Alternatively, protein mass spectrometry may be used to identify orvalidate the presence of mutated peptides bound to MHC proteins on tumorcells. Peptides can be acid-eluted from tumor cells or from HLAmolecules that are immunoprecipitated from tumor, and then identifiedusing mass spectrometry.

Neoantigenic Peptides

The invention further includes isolated peptides that comprise the tumorspecific mutations identified by the methods of the invention, peptidesthat comprise know tumor specific mutations, and mutant polypeptides orfragments thereof identified by the method of the invention. Thesepeptides and polypeptides are referred to herein as “neoantigenicpeptides” or “neoantigenic polypeptides”. The term “peptide” is usedinterchangeably with “mutant peptide” and “neoantigenic peptide” in thepresent specification to designate a series of residues, typicallyL-amino acids, connected one to the other, typically by peptide bondsbetween the α-amino and carboxyl groups of adjacent amino acids.Similarly, the term “polypeptide” is used interchangeably with “mutantpolypeptide” and “neoantigenic polypeptide” in the present specificationto designate a series of residues, typically L-amino acids, connectedone to the other, typically by peptide bonds between the α-amino andcarboxyl groups of adjacent amino acids. The polypeptides or peptidescan be a variety of lengths, either in their neutral (uncharged) formsor in forms which are salts, and either free of modifications such asglycosylation, side chain oxidation, or phosphorylation or containingthese modifications, subject to the condition that the modification notdestroy the biological activity of the polypeptides as herein described.

In certain embodiments the size of the at least one neoantigenic peptidemolecule may comprise, but is not limited to, about 5, about 6, about 7,about 8, about 9, about 10, about 11, about 12, about 13, about 14,about 15, about 16, about 17, about 18, about 19, about 20, about 21,about 22, about 23, about 24, about 25, about 26, about 27, about 28,about 29, about 30, about 31, about 32, about 33, about 34, about 35,about 36, about 37, about 38, about 39, about 40, about 41, about 42,about 43, about 44, about 45, about 46, about 47, about 48, about 49,about 50, about 60, about 70, about 80, about 90, about 100, about 110,about 120 or greater amino molecule residues, and any range derivabletherein. In specific embodiments the neoantigenic peptide molecules areequal to or less than 50 amino acids.

In some embodiments the particular neoantigenic peptides andpolypeptides of the invention are: for MHC Class I 13 residues or lessin length and usually consist of between about 8 and about 11 residues,particularly 9 or 10 residues; for MHC Class II, 15-24 residues.

A longer peptide may be designed in several ways. In one case, whenHLA-binding peptides are predicted or known, a longer peptide couldconsist of either: (1) individual binding peptides with an extensions of2-5 amino acids toward the N- and C-terminus of each corresponding geneproduct; (2) a concatenatation of some or all of the binding peptideswith extended sequences for each. In another case, when sequencingreveals a long (>10 residues) neoepitope sequence present in the tumor(e.g. due to a frameshift, read-through or intron inclusion that leadsto a novel peptide sequence), a longer peptide would consist of: (3) theentire stretch of novel tumor-specific amino acids—thus bypassing theneed for computational prediction or in vitro testing of peptide bindingto HLA proteins. In both cases, use of a longer peptide allowsendogenous processing by patient cells and may lead to more effectiveantigen presentation and induction of T cell responses.

The neoantigenic peptides and polypeptides bind an HLA protein. In someaspect the neoantigenic peptides and polypeptides binds an HLA proteinwith greater affinity than a wild-type peptide. The neoantigenic peptideor polypeptide has an IC50 of at least less than 5000 nM, at least lessthan 500 nM, at least less then 250 nM, at least less than 200 nM, atleast less than 150 nM, at least less than 100 nM, at least less than 50nM or less.

The neoantigenic peptides and polypeptides does not induce an autoimmuneresponse and/or invoke immunological tolerance when administered to asubject.

The invention also provides compositions comprising at least two or moreneoantigenic peptides. In some embodiments the composition contains atleast two distint peptides. Preferably, the at least two distintpeptides are derived from the same polypeptide. By distint polypeptidesis meant that the peptide vary by length, amino acid sequence or both.The peptides are derived from any polypeptide know to or have been foundto by the methods of the invention to contain a tumor specific mutation.Suitable polypeptides from which the neoantigenic peptides may bederived can be found for example at the COSMIC database. COSMIC curatescomprehensive information on somatic mutations in human cancer. Thepeptide contains the tumor specific mutation. In some aspects the tumorspecific mutation is a driver mutation for a particular cancer type. Insome aspects, the peptides are derived from a SF3B1 polypeptide, a MYD88polypeptide, a TP53 polypeptide, an ATM polypeptide, an Abl polypeptide,A FBXW7 polypeptide, a DDX3X polypeptide, a MAPK1 polypeptide of a GNB1polypeptide.

By a SF3B1 peptide is meant that the peptide contains a portion of aSF3B1 polypeptide. Preferably, a SF3B1 peptide includes either leucineat amino acid position 625; a histidine at amino acid position 626; aglutamic acid at amino acid position 700; an aspartic acid at amino acidposition 742; or an arginine at amino acid position 903, when numberedin accordance with wild-type SF3B1. A wild type SF3B1 is shown in TableA (SEQ ID NO: 1).

TABLE A Wild Type SF3B1 (SEQ ID NO: 1)makiakthedieaqireiqgkkaaldeaqgvgldstgyydgeiyggsdsrfagyvtsiaateledddddyssstsllgqkkpgyhapvallndipgstegydpfaehrppkiadredeykkhrrtmiisperldpfadggktpdpkmnartymdvmreghltkeereirgqlaekakagelkvvngaaasqppskrkrrwdqtadqtpgatpkklsswdgaetpghtpslrwdetpgrakgsetpgatpgskiwdptpshtpagaatpgrgdtpghatpghggatssarknrwdetpkterdtpqhgsgwaetprtdrggdsigetptpgaskrksrwdetpasqmggstpvltpgktpigtpamnmatptpghimsmtpeglqawrwereidernrplsdeeldamfpegykvlpppagyvpirtparkltatptplggmtgfhmqtedrtmksvndqpsgnlpflkpddigyfdkllvdvdestlspeegkerkimklllkikngtppmrkaalrqitdkarefgagplfnqilpllmsptledgerhllvkvidrilyklddlvrpyvhkilvviepllidedyyarvegreiisnlakaaglatmistmrpdidnmdeyvrnttarafavvasalgipsllpflkavckskkswgarhtgikivqqiailmgcailphlrslveiiehqlvdeqqkvrtisalaiaalaeaatpygiesfdsvikplwkqirqhrgkqlaaflkaigyliplmdaeyanyytrevmlilirefgspdeemkkivlkvvkqccqtdgveanyikteilppffkhfwqhrmaldrrnyrglvdttvelankvgaaeiisrivddlkdeaegyrkmvmetiekimgnlgaadidhkleeqlidgilyafqeqttedsvmlngfgtvvnalgkrvkpylpqicgtvlwrlnnksakvrqgaadlisrtavvmktcqeeklmghlgvvlyeylgeeypevlgsilgalkaivnvigmhkmtppikdllprltpilknrhekvqencidlvqriadrgaeyvsarewmricfellellkahkkairratvntfgyiakaigphdvlatllnnlkyqergnrvcttvaiaivaetcspftvlpalmneyrvpelnvgnqvlkslsflfeyigemgkdyiyavtplledalmdrdlvhrqtasavvqhmslgvygfgcedslnhllnyvwpnvfetsphvigavmgaleglrvaigpcrmlqyclqglfhparkvrdvywkiynsiyigsqdaliahypriynddkntyiryel dyil

By a MYD88 peptide is meant that the peptide contains a portion of aMYD88 polypeptide. Preferably, a MYD88 peptide includes either athreonine at amino acid position 232; a leucine at amino acid position258; or a proline at amino acid position 265, when numbered inaccordance with wild-type MYD88 when numbered in accordance withwild-type MYD88. A wild type MYD88 is shown in Table B (SEQ ID NO:2).

TABLE B Wild Type MYD88 (SEQ ID NO: 2)mrpdraeapgppamaaggpgagsaapvsstsslplaalnmrvrrrlslflnvrtqvaadwtalaeemdfeyleirqletqadptgrlldawqgrpgasvgrllelltklgrddvllelgpsieedcqkyilkqqqeeaekplgvaavdssvprtaelagittlddplghmperfdaficycpsdiqfvqemirqleqtnyrlklcvsdrdvlpgtcvwsiaseliekrcrrmvvvvsddylqskecdfqtkfalslspgahqkrlipikykamkkefpsilrfitvcdytnpctkswfwt rlakalslp

By a TP53 peptide is meant that the peptide contains a portion of a TP53polypeptide. Preferably, a TP53 peptide includes either an arginine atamino acid position 111; an arginine at amino acid position 215; aserine at amino acid position 238; a glutamine at amino acid position248; a phenylalanine at amino acid position 255; a cysteine at aminoacid position 273 or an asparagine at amino acid position 281, whennumbered in accordance with wild-type TP53. A wild type TP53 is shown inTable C (SEQ ID NO:3).

TABLE C Wild Type TP53 (SEQ ID NO:3)meepqsdpsvepplsqetfsdlwkllpennvlsplpsqamddlmlspddieqwftedpgpdeaprmpeaappvapapaaptpaapapapswplsssvpsqktyqgsygfrlgflhsgtaksvtctyspalnkmfcqlaktcpvqlwvdstpppgtrvramaiykqsqhmtevvrrcphhercsdsdglappqhlirvegnlrveylddrntfrhsvvvpyeppevgsdcttihynymcnsscmggmnrrpiltiitledssgnllgrnsfevrvcacpgrdrrteeenlrkkgephhelppgstkralpnntssspqpkkkpldgeyftlqirgrerfemfrelnealelkdaqagkepggsrahsshlkskkgqstsrhkklmfktegpdsd

-   -   By an ATM peptide is meant that the peptide contains a portion        of a SF3B1 polypeptide. Preferably, a ATM peptide includes        either a phenylalanine at amino acid position 1252; an arginine        at amino acid position 2038; a histidine at amino acid position        2522; or a cysteine at amino acid position 2954, when numbered        in accordance with wild-type ATM.        A wild type ATM is shown in Table D (SEQ ID NO:4).

Table D Wild Type ATM (SEQ ID NO: 4)mslvlndlliccrqlehdraterkkevekfkrlirdpetikhldrhsdskqgkylnwdavfrflakyiqketeclriakpnvsastqasrqkkmqeisslvkyfikcanrraprlkcqellnyimdtvkdssngaiygadcsnillkdilsvrkywceisqqqwlelfsvyfrlylkpsqdvhrvlvariihavtkgccsqtdglnskfldffskaiqcarqeksssglnhilaaltiflktlavnfrirvcelgdeilptllyiwtqhrlndslkeviielfqlqiyihhpkgaktqekgayestkwrsilynlydllvneishigsrgkyssgfrniavkenlielmadichqvfnedtrsleisqsytttqressdysvpckrkkielgwevikdhlqksqndfdlvpwlqiatqliskypaslpncelspllmilsqllpqqrhgertpyvlrcltevalcqdkrsnlessqksdllklwnkiwcitfrgisseqiqaenfgllgaiiqgslvevdrefwklftgsacrpscpavccltlalttsivpgtvkmgieqnmcevnrsfslkesimkwllfyqlegdlenstevppilhsnfphlvlekilvsltmknckaamnffqsvpecehhqkdkeelsfseveelflqllfdkmdfltivrecgiekhqssigfsvhqnlkesldrcllglseqllnnysseitnsetlvrcsrllvgvlgcycymgviaeeeaykselfqkakslmqcagesitlfknktneefrigslrnmmqlctrclsnctkkspnkiasgfflrlltsklmndiadickslasfikkpfdrgevesmeddtngnlmevedqssmnlfndypdssvsdanepgesqstigainplaeeylskqdllfldmlkflclcvttaqtntvsfraadirrkllmlidsstleptkslhlhmylmllkelpgeeyplpmedvlellkplsnvcslyrrdqdvcktilnhvlhvvknlgqsnmdsentrdaqgqfltvigafwhltkerkyifsvrmalvnclktlleadpyskwailnvmgkdfpvnevftqfladnhhqvrmlaaesinrlfqdtkgdssrllkalplklqqtafenaylkaqegmremshsaenpetldeiynrksvlltliavvlscspicekqalfalcksvkenglephlvkkviekvsetfgyrrledfmashldylvlewlnlqdteynlssfpfillnytniedfyrscykvliphlvirshfdevksianqiqedwkslltdcfpkilvnilpyfayegtrdsgmaqqretatkvydmlksenllgkqidhlfisnlpeivvellmtlhepanssasqstdlcdfsgdldpapnpphfpshvikatfayisnchktklksileilskspdsyqkillaiceqaaetnnvykkhrilkiyhlfvslllkdiksglggawafvlrdviytlihyinqrpscimdvslrsfslccdllsqvcqtavtyckdalenhlhvivgtliplvyeqvevqkqvldllkylvidnkdnenlyitiklldpfpdhvvfkdlritqqkikysrgpfslleeinhflsvsvydalpltrleglkdlrrqlelhkdqmvdimrasqdnpqdgimvklvvnllqlskmainhtgekevleavgsclgevgpidfstiaiqhskdasytkalklfedkelqwtfimltylnntlvedcvkvrsaavtclknilatktghsfweiykmttdpmlaylqpfrtsrkkflevprfdkenpfeglddinlwiplsenhdiwiktltcafldsggtkceilqllkpmcevktdfcqtvlpylihdillqdtneswrnllsthvqgfftsclrhfsqtsrsttpanldsesehffrccldkksqrtmlavvdymrrqkrpssgtifndafwldlnylevakvaqscaahftallyaeiyadkksmddqekrslafeegsqsllisslsekskeetgislqdllleiyrsigepdslygcgggkmlqpitrlrtyeheamwgkalvtydletaipsstrqagiiqalqnlglchilsvylkgldyenkdwcpeleelhyqaawrnmqwdhctsvskevegtsyheslynalqslrdrefstfyeslkyarvkeveemckrslesvyslyptlsrlqaigelesigelfsrsvthrqlsevyikwqkhsqllkdsdfsfqepimalrtvileilmekemdnsqrecikdiltkhlvelsilartfkntqlperaifqikqynsvscgvsewqleeaqvfwakkeqslalsilkqmikkldascaannpslkltyteclrvcgnwlaetclenpavimqtylekavevagnydgessdelrngkmkaflslarfsdtqyqrienymkssefenkqallkrakeevgilrehkiqtnrytvkvqreleldeialralkedrkrflckavenyincllsgeehdmwvfrlcslwlensgvsevngmmkrdgmkiptykflplmyqlaarmgtkmmgglgfhevlnnlisrismdhphhtlfiilalananrdefltkpevarrsritknvpkqssqldedrteaanriictirsrrpqmvrsvealcdayiilanldatqwktqrkginipadqpitklknledvvvptmeikvdhtgeygnlvtiqsfkaefrlaggvnlpkiidcvgsdgkerrqlvkgrddlrqdavmqqvfqmcntllqrntetrkrkltictykvvplsqrsgvlewctgtvpigeflvnnedgahkryrpndfsafqcqkkmmevqkksfeekyevfmdvcqnfqpvfryfcmekfldpaiwfekrlaytrsvatssivgyilglgdrhvqnilineqsaelvhidlgvafeqgkilptpetvpfrltrdivdgmgitgvegvfrrccektmevmrnsqetlltivevllydplfdwtmnplkalylqqrpedetelhptlnaddaeckrnlsdidqsfnkvaervlmrlqeklkgveegtvlsvggqvnlliqqaidpknlsrlfp gwkawv

By an Abl peptide is meant that the peptide contains a portion of an Ablpolypeptide. Preferably, a Bcr-abl peptide includes a valine at aminoacid position 244; a valine at amino acid position 248; a glutamic acidat amino acid position 250; an alanine at amino acid position 250; ahistidine at amino acid position 252; an arginine at amino acid position252; a phenylalanine at amino acid position 253; a histidine at aminoacid position 253; a lysine at amino acid position 255; a valine atamino acid position 255; a glycine at amino acid position 276; anisoleucine at amino acid position 315; an asparagine at amino acidposition 315; a leucine at amino acid position 317; a threonine at aminoacid position 343; a threonine at amino acid position 351; a glycine atamino acid position 355; a valine at amino acid position 359; an alanineat amino acid position 359; an isoleucine at amino acid position 379; aleucine at amino acid position 382; a methionine at amino acid position387; a proline at amino acid position 396; an arginine at amino acidposition 396; a tyrosine at amino acid position 417; or a serine atamino acid position 486, when numbered in accordance with wild-type Abl.A wild type Abl is shown in Table E (SEQ ID NO:5).

TABLE E Wild Type Ab1 (SEQ ID NO: 5)MLEICLKLVGCKSKKGLSSSSSCYLEEALQRPVASDFEPQGLSEAARWNSKENLLAGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLSSGINGSFLVRESESSPGQRSISLRYEGRVYHYRINTASDGKLYVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVEEFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRLMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRTSRRAAEHRDTTDVPEMPHSKGQGESDPLDHEPAVSPLLPRKERGPPEGGLNEDERLLPKDKKTNLFSALIKKKKKTAPTPPKRSSSFREMDGQPERRGAGEEEGRDISNGALAFTPLDTADPAKSPKPSNGAGVPNGALRESGGSGFRSPHLWKKSSTLTSSRLATGEEEGGGSSSKRFLRSCSASCVPHGAKDTEWRSVTLPRDLQSTGRQFDSSTFGGHKSEKPALPRKRAGENRSDQVTRGTVTPPPRLVKKNEEAADEVFKDIMESSPGSSPPNLTPKPLRRQVTVAPASGLPHKEEAGKGSALGTPAAAEPVTPTSKAGSGAPGGTSKGPAEESRVRRHKHSSESPGRDKGKLSRLKPAPPPPPAASAGKAGGKPSQSPSQEAAGEAVLGAKTKATSLVDAVNSDAAKPSQPGEGLKKPVLPATPKPQSAKPSGTPISPAPVPSTLPSASSALAGDQPSSTAFIPLISTRVSLRKTRQPPERIASGAITKGVVIDSTEALCLAISRNSEQMASKSAVLEAGKNLYTFCVSYVDSIQQMRNKFAFREAINKLENNLRELQICPATAGSGPAATQDFSKLLSSVKEISDIVQR

By a FBXW7 peptide is meant that the peptide contains a portion of aFBXW7 polypeptide. Preferably, a FBXW7 peptide includes either a leucineat amino acid position 280; a histidine at amino acid position 465; acysteine at amino acid position 505; or a glutamic acid at amino acidposition 597, when numbered in accordance with wild-type FBXW7. A wildtype FBXW7 is shown in Table F (SEQ ID N06).

TABLE F Wild Type FBXW7 (SEQ ID NO: 6)mnqellsvgskrrrtqgslrgnpsssqvdeegmnrvveeeqqqqlrqqeeehtarngevvgveprpggqndsqqgqleennnrfisvdedssgngeeqeedeehagegdeedeeeeemdqesddfdgsddssredehthtnsvtnsssivdlpvhqlsspfytkttkmkrkldhqsevrsfslgkkpckvseytsttglvpcsatpttfgdlraangqgqqrrritsvqpptglgewlkmfqswsgpekilaldelidsceptqvkhmmqviepqfqrdfisllpkelalyvlsflepkdllqaaqtcrywrilaednllwrekckeegideplhikrrkvikpgfihspwksayirqhridtnwrrgelkspkvlkghddhvitclqfcgnrivsgsddntlkvwsavtgkclrtlvqhtggvwssqmrdniiisgstdrtlkvwnaetgecihtlyghtstvrcmhlhekrvvsgsrdatlrvwdietgqclhvlmghvaavrcvqydgrrvvsgaydfmvkvwdpetetclhtlqghtnrvyslqfdgihvvsgsldtsirvwdvetgncihtltghqsltsgmelkdnilvsqnadstvkiwdiktgqclqtlqgpnkhqsavtclqfnknfvitssddgtvklwdlktgefirnlvtlesggsggvvwrirasntklvcavgsrngteetkllvl dfdvdmk

By a DDX3X peptide is meant that the peptide contains a portion of aDDX3X polypeptide. A DDX3X peptide is a peptide that is the result of amissence mutation at amino acid position 24; a splice site at amino acidposition 342 or a frame shift at amino acid position 410 when numberedin accordance with wild-type DDX3X. A wild type DDX3X is shown in TableG (SEQ ID NO:7).

TABLE F Wild Type DDX3X (SEQ ID NO: 7)mshvavenalgldqqfagldlnssdnqsggstaskgryipphlrnreatkgfydkdssgwssskdkdayssfgsrsdsrgkssffsdrgsgsrgrfddrgrsdydgigsrgdrsgfgkferggnsrwcdksdeddwskplppserleqelfsggntginfekyddipveatgnncpphiesfsdvemgeiimgnieltrytrptpvqkhaipiikekrdlmacaqtgsgktaafllpilsqiysdgpgealramkengrygrrlqypislvlaptrelavqiyeearkfsyrsrvrpcvvyggadigqqirdlergchllvatpgrlvdmmergikgldfckylvldeadrmldmgfepqirriveqdtmppkgvrhtmmfsatfpkeiqmlardfldeyiflavgrvgstsenitqkvvwveesdkrsflldllnatgkdsltlvfvetkkgadsledflyhegyactsihgdrsqrdreealhqfrsgkspilvatavaargldisnvkhvinfdlpsdieeyvhrigrtgrvgnlglatsffnerninitkdlldllveakqevpswlenmayeggykgssrgrskssrfsggfgardyrqssgassssfsssrasssrsgggghgssrgfggggyggfynsdgygg nynsqgvdwwgn

By a MAPK1 peptide is meant that the peptide contains a portion of aMAPK polypeptide. Preferably, a MAPK1 peptide includes either anasparagine at amino acid position 162; a glycine at amino acid position291; or a phenylalanine at amino acid position 316, when numbered inaccordance with wild-type MAPK1. A wild type MAPK1 is shown in Table H(SEQ ID NO:8).

Table F Wild Type MAPKI (SEQ ID NO: 8)maaaaaagagpemvrgqvfdvgprytnlsyigegaygmvcsaydnvnkvrvaikkispfehqtycqrtlreikillrfrheniigindiiraptieqmkdvyivqdlmetdlykllktqhlsndhicyflyqilrglkyihsanvlhrdlkpsnlllnttcdlkicdfglarvadpdhdhtqflteyvatrwyrapeimlnskgytksidiwsvgcilaemlsnrpifpqkhyldglnhilgilgspsgedlnciinlkarnyllslphknkvpwnrlfpnadskaldlldkmltfnphkrieveqalahpyleqyydpsdepiaeapfkfdmelddlpkeklkelifee tarfqpgyrs

By a GNB1 peptide is meant that the peptide contains a portion of a GNB1polypeptide. Preferably, a GNB1 peptide includes a threonine at aminoacid position 180, when numbered in accordance with wild-type GNB1. Awild type GNB1 is shown in Table I (SEQ ID NO9).

TABLE I Wild Type GNB1 (SEQ ID NO: 9)mseldqlrqeaeqlknqirdarkacadatlsqitnnidpvgriqmrtrrtlrghlakiyamhwgtdsrllvsasqdgkliiwdsyttnkvhaiplrsswvmtcayapsgnyvacggldnicsiynlktregnvrvsrelaghtgylsccrflddnqivtssgdttcalwdietgqqtttftghtgdvmslslapdtrlfysqacdasaklwdvregmcrqtftghesdinaicffpngnafatgsddatcrlfdlradqelmtyshdniicgitsvsfsksgrlllagyddfncnvwdalkadragvlaghdnrvsclgvtddamavatgswdsflkiwn

Neoantigenic peptides and polypeptides having the desired activity maybe modified as necessary to provide certain desired attributes, e.g.improved pharmacological characteristics, while increasing or at leastretaining substantially all of the biological activity of the unmodifiedpeptide to bind the desired MHC molecule and activate the appropriate Tcell. For instance, the neoantigenic peptide and polypeptides may besubject to various changes, such as substitutions, either conservativeor non-conservative, where such changes might provide for certainadvantages in their use, such as improved MHC binding. By conservativesubstitutions is meant replacing an amino acid residue with anotherwhich is biologically and/or chemically similar, e.g., one hydrophobicresidue for another, or one polar residue for another. The substitutionsinclude combinations such as Gly, Ala; Val, Ile, Leu, Met; Asp, Glu;Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. The effect of single aminoacid substitutions may also be probed using D-amino acids. Suchmodifications may be made using well known peptide synthesis procedures,as described in e.g., Merrifield. Science 232:341-347 (1986), Barany &Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., AcademicPress), pp. 1-284 (1979); and Stewart & Young, Solid Phase PeptideSynthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).

The neoantigenic peptide and polypeptides can also be modified byextending or decreasing the compound's amino acid sequence, e.g., by theaddition or deletion of amino acids. The peptides, polypeptides oranalogs can also be modified by altering the order or composition ofcertain residues, it being readily appreciated that certain amino acidresidues essential for biological activity, e.g., those at criticalcontact sites or conserved residues, may generally not be alteredwithout an adverse effect on biological activity. The non-critical aminoacids need not be limited to those naturally occurring in proteins, suchas L-α-amino acids, or their D-isomers, but may include non-naturalamino acids as well, such as β-γ-δ-amino acids, as well as manyderivatives of L-α-amino acids.

Typically, a series of peptides with single amino acid substitutions areemployed to determine the effect of electrostatic charge,hydrophobicity, etc. on binding. For instance, a series of positivelycharged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acidsubstitutions are made along the length of the peptide revealingdifferent patterns of sensitivity towards various MHC molecules and Tcell receptors. In addition, multiple substitutions using small,relatively neutral moieties such as Ala, Gly. Pro, or similar residuesmay be employed. The substitutions may be homo-oligomers orhetero-oligomers. The number and types of residues which are substitutedor added depend on the spacing necessary between essential contactpoints and certain functional attributes which are sought (e.g.,hydrophobicity versus hydrophilicity). Increased binding affinity for anMHC molecule or T cell receptor may also be achieved by suchsubstitutions, compared to the affinity of the parent peptide. In anyevent, such substitutions should employ amino acid residues or othermolecular fragments chosen to avoid, for example, steric and chargeinterference which might disrupt binding.

Amino acid substitutions are typically of single residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final peptide. Substitutional variants are thosein which at least one residue of a peptide has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with the following Table when it is desired tofinely modulate the characteristics of the peptide.

Original Residue Exemplary Substitution Ala Ser Arg Lys, His Asn Gln AspGlu Cys Ser Gln Asn Glu Asp Gly Pro His Lys; Arg Ile Leu; Val Leu Ile;Val Lys Arg; His Met Leu; Ile Phe Tyr; Trp Ser Thr Thr Ser Trp Tyr; PheTyr Trp; Phe Val Ile; Leu Pro Gly

Substantial changes in function (e.g., affinity for MHC molecules or Tcell receptors) are made by selecting substitutions that are lessconservative than those in above Table, i.e., selecting residues thatdiffer more significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, forexample as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in peptide properties will be those in which (a)hydrophilic residue, e.g. seryl, is substituted for (or by) ahydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a residue having an electropositive side chain, e.g., lysl,arginyl, or histidyl, is substituted for (or by) an electronegativeresidue, e.g. glutamyl or aspartyl; or (c) a residue having a bulky sidechain, e.g. phenylalanine, is substituted for (or by) one not having aside chain, e.g., glycine.

The peptides and polypeptides may also comprise isosteres of two or moreresidues in the neoantigenic peptide or polypepeptides. An isostere asdefined here is a sequence of two or more residues that can besubstituted for a second sequence because the steric conformation of thefirst sequence fits a binding site specific for the second sequence. Theterm specifically includes peptide backbone modifications well known tothose skilled in the art. Such modifications include modifications ofthe amide nitrogen, the α-carbon, amide carbonyl, complete replacementof the amide bond, extensions, deletions or backbone crosslinks. See,generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Vol. VII (Weinstein ed., 1983).

Modifications of peptides and polypeptides with various amino acidmimetics or unnatural amino acids are particularly useful in increasingthe stability of the peptide and polypeptide in vivo. Stability can beassayed in a number of ways. For instance, peptidases and variousbiological media, such as human plasma and serum, have been used to teststability. See, e.g., Verhoef et al., Eur. J. Drug Metab Pharmacokin.11:291-302 (1986). Half life of the peptides of the present invention isconveniently determined using a 25% human serum (v/v) assay. Theprotocol is generally as follows. Pooled human serum (Type AB, non-heatinactivated) is delipidated by centrifugation before use. The serum isthen diluted to 25% with RPMI tissue culture media and used to testpeptide stability. At predetermined time intervals a small amount ofreaction solution is removed and added to either 6% aqueoustrichloracetic acid or ethanol. The cloudy reaction sample is cooled (4°C.) for 15 minutes and then spun to pellet the precipitated serumproteins. The presence of the peptides is then determined byreversed-phase HPLC using stability-specific chromatography conditions.

The peptides and polypeptides may be modified to provide desiredattributes other than improved serum half life. For instance, theability of the peptides to induce CTL activity can be enhanced bylinkage to a sequence which contains at least one epitope that iscapable of inducing a T helper cell response. Particularly preferredimmunogenic peptides/T helper conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues. Alternatively, the peptide may be linked to the Thelper peptide without a spacer.

The neoantigenic peptide may be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of thepeptide. The amino terminus of either the neoantigenic peptide or the Thelper peptide may be acylated. Exemplary T helper peptides includetetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite382-398 and 378-389.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases located at theNational Institutes of Health website. The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In a further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a neoantigenic peptide of the invention. Thepolynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA, either single-and/or double-stranded, or native or stabilized forms ofpolynucleotides, such as e.g. polynucleotides with a phosphorothiatebackbone, or combinations thereof and it may or may not contain intronsso long as it codes for the peptide. Of course, only peptides thatcontain naturally occurring amino acid residues joined by naturallyoccurring peptide bonds are encodable by a polynucleotide. A stillfurther aspect of the invention provides an expression vector capable ofexpressing a polypeptide according to the invention. Expression vectorsfor different cell types are well known in the art and can be selectedwithout undue experimentation. Generally, the DNA is inserted into anexpression vector, such as a plasmid, in proper orientation and correctreading frame for expression. If necessary, the DNA may be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host through standard techniques. Guidance canbe found e.g. in Sambrook et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Vaccine Compositions

The present invention is directed to an immunogenic composition, e.g., avaccine composition capable of raising a specific T-cell response. Thevaccine composition comprises mutant peptides and mutant polypeptidescorresponding to tumor specific neoantigens identified by the methodsdescribed herein.

A person skilled in the art will be able to select preferred peptides,polypeptide or combination of therof by testing, for example, thegeneration of T-cells in vitro as well as their efficiency and overallpresence, the proliferation, affinity and expansion of certain T-cellsfor certain peptides, and the functionality of the T-cells, e.g. byanalyzing the IFN-γ production or tumor killing by T-cells. Usually, themost efficient peptides are then combined as a vaccine.

A suitable vaccine will preferably contain between 1 and 20 peptides,more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 different peptides, further preferred 6, 7, 8, 9, 10 11,12, 13, or 14 different peptides, and most preferably 12, 13 or 14different peptides.

In one embodiment of the present invention the different peptides and/orpolypeptides are selected so that one vaccine composition comprisespeptides and/or polypeptides capable of associating with different MHCmolecules, such as different MHC class I molecule. Preferably, onevaccine composition comprises peptides and/or polypeptides capable ofassociating with the most frequently occurring MHC class I molecules.Hence vaccine compositions according to the invention comprisesdifferent fragments capable of associating with at least 2 preferred,more preferably at least 3 preferred, even more preferably at least 4preferred MHC class I molecules.

The vaccine composition is capable of raising a specific cytotoxicT-cells response and/or a specific helper T-cell response.

The vaccine composition can further comprise an adjuvant and/or acarrier. Examples of useful adjuvants and carriers are given hereinbelow. The peptides and/or polypeptides in the composition can beassociated with a carrier such as e.g. a protein or anantigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into the vaccine compositionincreases or otherwise modifies the immune response to the mutantpeptide. Carriers are scaffold structures, for example a polypeptide ora polysaccharide, to which the neoantigenic peptides, is capable ofbeing associated. Optionally, adjuvants are conjugated covalently ornon-covalently to the peptides or polypeptides of the invention.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedreaction, or reduction in disease symptoms. For example, an increase inhumoral immunity is typically manifested by a significant increase inthe titer of antibodies raised to the antigen, and an increase in T-cellactivity is typically manifested in increased cell proliferation, orcellular cytotoxicity, or cytokine secretion. An adjuvant may also alteran immune response, for example, by changing a primarily humoral or Thresponse into a primarily cellular, or Th response.

Suitable adjuvants include, but are not limited to 1018 ISS, aluminiumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF,IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V. Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (AquilaBiotech, Worcester, Mass., USA) which is derived from saponin,mycobacterial extracts and synthetic bacterial cell wall mimics, andother proprietary adjuvants such as Ribi's Detox. Quil or Superfos.Adjuvants such as incomplete Freund's or GM-CSF are preferred. Severalimmunological adjuvants (e.g., MF59) specific for dendritic cells andtheir preparation have been described previously (Dupuis M, et al., CellImmunol. 1998; 186(1):18-27; Allison A C; Dev Biol Stand. 1998;92:3-11). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly, it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T-cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nano particles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enabled the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun.2006, 471-484). U.S. Pat. No. 6,406,705 B1 describes the combined use ofCpG oligonucleotides, non-nucleic acid adjuvants and an antigen toinduce an antigen-specific immune response. A commercially available CpGTLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, GERMANY), which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples of useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), Poly(I:C) (e.g. polyi:CI2U),non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan without undue experimentation. Additional adjuvantsinclude colony-stimulating factors, such as Granulocyte MacrophageColony Stimulating Factor (GM-CSF, sargramostim).

A vaccine composition according to the present invention may comprisemore than one different adjuvants. Furthermore, the inventionencompasses a therapeutic composition comprising any adjuvant substanceincluding any of the above or combinations thereof. It is alsocontemplated that the peptide or polypeptide, and the adjuvant can beadministered separately in any appropriate sequence.

A carrier may be present independently of an adjuvant. The function of acarrier can for example be to increase the molecular weight of inparticular mutant in order to increase their activity or immunogenicity,to confer stability, to increase the biological activity, or to increaseserum half-life. Furthermore, a carrier may aid presenting peptides toT-cells. The carrier may be any suitable carrier known to the personskilled in the art, for example a protein or an antigen presenting cell.A carrier protein could be but is not limited to keyhole limpethemocyanin, serum proteins such as transferrin, bovine serum albumin,human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, orhormones, such as insulin or palmitic acid. For immunization of humans,the carrier must be a physiologically acceptable carrier acceptable tohumans and safe. However, tetanus toxoid and/or diptheria toxoid aresuitable carriers in one embodiment of the invention. Alternatively, thecarrier may be dextrans for example sepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is only possible if atrimeric complex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodimentsthe vaccine composition according to the present invention additionallycontains at least one antigen presenting cell.

The antigen-presenting cell (or stimulator cell) typically has an MHCclass I or II molecule on its surface, and in one embodiment issubstantially incapable of itself loading the MHC class I or II moleculewith the selected antigen. As is described in more detail below, the MHCclass I or II molecule may readily be loaded with the selected antigenin vitro.

Preferably, the antigen presenting cells are dendritic cells. Suitably,the dendritic cells are autologous dendritic cells that are pulsed withthe neoantigenic peptide. The peptide may be any suitable peptide thatgives rise to an appropriate T-cell response. T-cell therapy usingautologous dendritic cells pulsed with peptides from a tumor associatedantigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380and Tjua et al. (1997) The Prostate 32, 272-278.

Thus, in one embodiment of the present invention the vaccine compositioncontaining at least one antigen presenting cell is pulsed or loaded withone or more peptides of the present invention. Alternatively, peripheralblood mononuclear cells (PBMCs) isolated from a patient may be loadedwith peptides ex vivo and injected back into the patient.

As an alternative the antigen presenting cell comprises an expressionconstruct encoding a peptide of the present invention. Thepolynucleotide may be any suitable polynucleotide and it is preferredthat it is capable of transducing the dendritic cell, thus resulting inthe presentation of a peptide and induction of immunity.

Therapeutic Methods

The invention further provides a method of inducing a tumor specificimmune response in a subject, vaccinating against a tumor, treating andor alleviating a symptom of cancer in a subject by administering thesubject a neoantigenic peptide or vaccine composition of the invention.

The subject has been diagnosed with cancer or is at risk of developingcancer. The subject has a imatinib resistant tumor. The subject is ahuman, dog, cat, horse or any animal in which a tumor specific immuneresponse is desired. The tumor is any solid tumor such as breast,ovarian, prostate, lung, kidney, gastric, colon, testicular, head andneck, pancreas, brain, melanoma, and other tumors of tissue organs andhematological tumors, such as lymphomas and leukemias, including acutemyelogenous leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, T cell lymphocytic leukemia, and B cell lymphomas.

The peptide or composition of the invention is administered in an amountsufficient to induce a CTL response.

In specific embodiments, the invention provides methods of treating animatinib resistant tumor by administering to a subject one or moreneoantigenic peptides that contain a bcr-abl mutation. In someembodiments the subject is HLA-A3. Bcr-abl mutations include for exampleT315I, E255K, M351T, Y253H, Q252H, F317L, F359V, G250E, Y253F, E355G.E255V, M244V, L248V, G250A, Q252R, D276G, T315N, M343T, F359A, V379I,F382L, L387M, H396P, H396R, S417Y, F486S.

The neoantigenic peptide, polypeptide or vaccine composition of theinvention can be administered alone or in combination with othertherapeutic agents. The therapeutic agent is for example, achemotherapeutic agent, radiation, or immunotherapy. Any suitabletherapeutic treatment for a particular cancer may be administered.Examples of chemotherapeutic agents include, but are not limited to,aldesleukin, altretamine, amifostine, asparaginase, bleomycin,capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin,cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin,docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide,filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron,hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan,lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate,metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole,ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine,rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,vinblastine, vincristine and vinorelbine tartrate. For prostate cancertreatment, a preferred chemotherapeutic agent with which anti-CTLA-4 canbe combined is paclitaxel (Taxol®).

In addition, the subject may be further administered ananti-immunosuppressive/immunostimulatory agent. For example, the subjectis further administered an anti-CTLA antibody or anti-PD-1 oranti-PD-L1. Blockade of CTLA-4 or PD-L1 by antibodies can enhance theimmune response to cancerous cells in the patient. In particular, CTLA-4blockade has been shown effective when following a vaccination protocol.

The optimum amount of each peptide to be included in the vaccinecomposition and the optimum dosing regimen can be determined by oneskilled in the art without undue experimentation. For example, thepeptide or its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. For example, doses of between 1 and 500 mg 50 μg and 1.5mg, preferably 125 μg to 500 μg, of peptide or DNA may be given and willdepend from the respective peptide or DNA. Doses of this range weresuccessfully used in previous trials (Brunsvig P F, et al., CancerImmunol Immunother. 2006; 55(12):1553-1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017). Other methods of administion of thevaccine composition are known to thoses skilled in the art.

The inventive pharmaceutical composition may be compiled so that theselection, number and/or amount of peptides present in the compositionis/are tissue, cancer, and/or patient-specific. For instance, the exactselection of peptides can be guided by expression patterns of the parentproteins in a given tissue to avoid side effects. The selection may bedependent on the specific type of cancer, the status of the disease,earlier treatment regimens, the immune status of the patient, and, ofcourse, the HLA-haplotype of the patient. Furthermore, the vaccineaccording to the invention can contain individualized components,according to personal needs of the particular patient. Examples includevarying the amounts of peptides according to the expression of therelated neoantigen in the particular patient, unwanted side-effects dueto personal allergies or other treatments, and adjustments for secondarytreatments following a first round or scheme of treatment.

For a composition to be used as a vaccine for cancer, peptides whoseendogenous parent proteins are expressed in high amounts in normaltissues will be avoided or be present in low amounts in the compositionof the invention. On the other hand, if it is known that the tumor of apatient expresses high amounts of a certain protein, the respectivepharmaceutical composition for treatment of this cancer may be presentin high amounts and/or more than one peptide specific for thisparticularly protein or pathway of this protein may be included.

Pharmaceutical compositions comprising the peptide of the invention maybe administered to an individual already suffering from cancer. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective CTL response to the tumorantigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range for the initial immunization(that is for therapeutic or prophylactic administration) from about 1.0μg to about 50,000 μg of peptide for a 70 kg patient, followed byboosting dosages or from about 1.0 μg to about 10,000 μg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient's response and condition by measuring specific CTL activity inthe patient's blood. It must be kept in mind that the peptide andcompositions of the present invention may generally be employed inserious disease states, that is, life-threatening or potentially lifethreatening situations, especially when the cancer has metastasized. Insuch cases, in view of the minimization of extraneous substances and therelative nontoxic nature of the peptide, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these peptide compositions.

For therapeutic use, administration should begin at the detection orsurgical removal of tumors. This is followed by boosting doses until atleast symptoms are substantially abated and for a period thereafter.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. Preferably, the pharmaceutical compositions areadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The composisitions may be administerdat the site of surgical exiscion to induce a local immune response tothe tumor. The invention provides compositions for parenteraladministration which comprise a solution of the peptides and vaccinecompositions are dissolved or suspended in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers may beused, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronicacid and the like. These compositions may be sterilized by conventional,well known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

The peptide of the invention may also be administered via liposomes,which target the peptides to a particular cells tissue, such as lymphoidtissue. Liposomes are also useful in increasing the half-life of thepeptides. Liposomes include emulsions, foams, micelles, insolublemonolayers, liquid crystals, phospholipid dispersions, lamellar layersand the like. In these preparations the peptide to be delivered isincorporated as part of a liposome, alone or in conjunction with amolecule which binds to, e.g., a receptor prevalent among lymphoidcells, such as monoclonal antibodies which bind to the CD45 antigen, orwith other therapeutic or immunogenic compositions. Thus, liposomesfilled with a desired peptide of the invention can be directed to thesite of lymphoid cells, where the liposomes then deliver the selectedtherapeutic/immunogenic peptide compositions. Liposomes for use in theinvention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728,4,837,028 and 5,019,369.

For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension containing a peptide may be administered intravenously,locally, topically, etc. in a dose which varies according to, interalia, the manner of administration, the peptide being delivered, and thestage of the disease being treated.

For solid compositions, conventional or nanoparticle nontoxic solidcarriers may be used which include, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.For oral administration, a pharmaceutically acceptable nontoxiccomposition is formed by incorporating any of the normally employedexcipients, such as those carriers previously listed, and generally10-95% of active ingredient, that is, one or more peptides of theinvention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included asdesired, as with, e.g., lecithin for intranasal delivery.

For therapeutic or immunization purposes, nucleic acids encoding thepeptide of the invention and optionally one or more of the peptidesdescribed herein can also be administered to the patient. A number ofmethods are conveniently used to deliver the nucleic acids to thepatient. For instance, the nucleic acid can be delivered directly, as“naked DNA”. This approach is described, for instance, in Wolff et al.,Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and5,589,466. The nucleic acids can also be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253.Particles comprised solely of DNA can be administered. Alternatively,DNA can be adhered to particles, such as gold particles.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in 9618372WOAWO 96/18372; 9324640WOAWO93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);5279833USARose U.S. Pat. No. 5,279,833; 9106309WOAWO 91/06309; andFelgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

The peptides and polypeptided of the invention can also be expressed byattenuated viral hosts, such as vaccinia or fowlpox. This approachinvolves the use of vaccinia virus as a vector to express nucleotidesequences that encode the peptide of the invention. Upon introductioninto an acutely or chronically infected host or into a noninfected host,the recombinant vaccinia virus expresses the immunogenic peptide, andthereby elicits a host CTL response. Vaccinia vectors and methods usefulin immunization protocols are described in, e.g., U.S. Pat. No.4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectorsare described in Stover et al. (Nature 351:456-460 (1991)). A widevariety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

A preferred means of administering nucleic acids encoding the peptide ofthe invention uses minigene constructs encoding multiple epitopes. Tocreate a DNA sequence encoding the selected CTL epitopes (minigene) forexpression in human cells, the amino acid sequences of the epitopes arereverse translated. A human codon usage table is used to guide the codonchoice for each amino acid. These epitope-encoding DNA sequences aredirectly adjoined, creating a continuous polypeptide sequence. Tooptimize expression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencethat could be reverse translated and included in the minigene sequenceinclude: helper T lymphocyte, epitopes, a leader (signal) sequence, andan endoplasmic reticulum retention signal. In addition, MHC presentationof CTL epitopes may be improved by including synthetic (e.g.poly-alanine) or naturally-occurring flanking sequences adjacent to theCTL epitopes.

The minigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the CTL epitope polypeptide,can then cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the artare included in the vector to ensure expression in the target cells.Several vector elements are required: a promoter with a down-streamcloning site for minigene insertion; a polyadenylation signal forefficient transcription termination; an E. coli origin of replication;and an E. coli selectable marker (e.g. ampicillin or kanamycinresistance). Numerous promoters can be used for this purpose, e.g., thehuman cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencescan also be considered for increasing minigene expression. It hasrecently been proposed that immunostimulatory sequences (ISSs or CpGs)play a role in the immunogenicity of DNA' vaccines. These sequencescould be included in the vector, outside the minigene coding sequence,if found to enhance immunogenicity.

In some embodiments, a bicistronic expression vector, to allowproduction of the minigene-encoded epitopes and a second proteinincluded to enhance or decrease immunogenicity can be used. Examples ofproteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF),cytokine-inducing molecules (e.g. LeIF) or costimulatory molecules.Helper (HTL) epitopes could be joined to intracellular targeting signalsand expressed separately from the CTL epitopes. This would allowdirection of the HTL epitopes to a cell compartment different than theCTL epitopes. If required, this could facilitate more efficient entry ofHTL epitopes into the MHC class II pathway, thereby improving CTLinduction. In contrast to CTL induction, specifically decreasing theimmune response by co-expression of immunosuppressive molecules (e.g.TGF-β) may be beneficial in certain diseases.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques may become available. As noted above,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and MHC class I presentation of minigene-encoded CTLepitopes. The plasmid DNA is introduced into a mammalian cell line thatis suitable as a target for standard CTL chromium release assays. Thetransfection method used will be dependent on the final formulation.Electroporation can be used for “naked” DNA, whereas cationic lipidsallow direct in vitro transfection. A plasmid expressing greenfluorescent protein (GFP) can be co-transfected to allow enrichment oftransfected cells using fluorescence activated cell sorting (FACS).These cells are then chromium-51 labeled and used as target cells forepitope-specific CTL lines. Cytolysis, detected by 51 Cr release,indicates production of MHC presentation of mini gene-encoded CTLepitopes.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanMHC molecules are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g. IM for DNA in PBS, IP forlipid-complexed DNA). Twenty-one days after immunization, splenocytesare harvested and restimulated for 1 week in the presence of peptidesencoding each epitope being tested. These effector cells (CTLs) areassayed for cytolysis of peptide-loaded, chromium-51 labeled targetcells using standard techniques. Lysis of target cells sensitized by MHCloading of peptides corresponding to minigene-encoded epitopesdemonstrates DNA vaccine function for in vivo induction of CTLs.

Peptides may be used to elicit CTL ex vivo, as well. The resulting CTL,can be used to treat chronic tumors in patients that do not respond toother conventional forms of therapy, or will not respond to a peptidevaccine approach of therapy. Ex vivo CTL responses to a particular tumorantigen are induced by incubating in tissue culture the patient's CTLprecursor cells (CTLp) together with a source of antigen-presentingcells (APC) and the appropriate peptide. After an appropriate incubationtime (typically 1-4 weeks), in which the CTLp are activated and matureand expand into effector CTL, the cells are infused back into thepatient, where they will destroy their specific target cell (i.e., atumor cell). In order to optimize the in vitro conditions for thegeneration of specific cytotoxic T cells, the culture of stimulatorcells is maintained in an appropriate serum-free medium.

Prior to incubation of the stimulator cells with the cells to beactivated, e.g., precursor CD8+ cells, an amount of antigenic peptide isadded to the stimulator cell culture, of sufficient quantity to becomeloaded onto the human Class I molecules to be expressed on the surfaceof the stimulator cells. In the present invention, a sufficient amountof peptide is an amount that will allow about 200, and preferably 200 ormore, human Class I MHC molecules loaded with peptide to be expressed onthe surface of each stimulator cell. Preferably, the stimulator cellsare incubated with >2 μg/ml peptide. For example, the stimular cells areincubates with >3, 4, 5, 10, 15, or more μg/ml peptide.

Resting or precursor CD8+ cells are then incubated in culture with theappropriate stimulator cells for a time period sufficient to activatethe CD8+ cells. Preferably, the CD8+ cells are activated in anantigen-specific manner. The ratio of resting or precursorCD8+(effector) cells to stimulator cells may vary from individual toindividual and may further depend upon variables such as the amenabilityof an individual's lymphocytes to culturing conditions and the natureand severity of the disease condition or other condition for which thewithin-described treatment modality is used. Preferably, however, thelymphocyte:stimulator cell ratio is in the range of about 30:1 to 300:1.The effector/stimulator culture may be maintained for as long a time asis necessary to stimulate a therapeutically useable or effective numberof CD8+ cells.

The induction of CTL in vitro requires the specific recognition ofpeptides that are bound to allele specific MHC class I molecules on APC.The number of specific MHC/peptide complexes per APC is crucial for thestimulation of CTL, particularly in primary immune responses. Whilesmall amounts of peptide/MHC complexes per cell are sufficient to rendera cell susceptible to lysis by CTL, or to stimulate a secondary CTLresponse, the successful activation of a CTL precursor (pCTL) duringprimary response requires a significantly higher number of MHC/peptidecomplexes. Peptide loading of empty major histocompatability complexmolecules on cells allows the induction of primary cytotoxic Tlymphocyte responses. Peptide loading of empty major histocompatabilitycomplex molecules on cells enables the induction of primary cytotoxic Tlymphocyte responses.

Since mutant cell lines do not exist for every human MHC allele, it isadvantageous to use a technique to remove endogenous MHC-associatedpeptides from the surface of APC, followed by loading the resultingempty MHC molecules with the immunogenic peptides of interest. The useof non-transformed (non-tumorigenic), noninfected cells, and preferably,autologous cells of patients as APC is desirable for the design of CTLinduction protocols directed towards development of ex vivo CTLtherapies. This application discloses methods for stripping theendogenous MHC-associated peptides from the surface of APC followed bythe loading of desired peptides.

A stable MHC class I molecule is a trimeric complex formed of thefollowing elements: 1) a peptide usually of 8-10 residues, 2) atransmembrane heavy polymorphic protein chain which bears thepeptide-binding site in its α1 and α2 domains, and 3) a non-covalentlyassociated non-polymorphic light chain, β2 microglobuiin. Removing thebound peptides and/or dissociating the β2 microglobulin from the complexrenders the MHC class I molecules nonfunctional and unstable, resultingin rapid degradation. All MHC class I molecules isolated from PBMCs haveendogenous peptides bound to them. Therefore, the first step is toremove all endogenous peptides bound to MHC class I molecules on the APCwithout causing their degradation before exogenous peptides can be addedto them.

Two possible ways to free up MHC class I molecules of bound peptidesinclude lowering the culture temperature from 37° C. to 26° C. overnightto destablize β2 microglobulin and stripping the endogenous peptidesfrom the cell using a mild acid treatment. The methods releasepreviously bound peptides into the extracellular environment allowingnew exogenous peptides to bind to the empty class I molecules. Thecold-temperature incubation method enables exogenous peptides to bindefficiently to the MHC complex, but requires an overnight incubation at26° C. which may slow the cell's metabolic rate. It is also likely thatcells not actively synthesizing MHC molecules (e.g., resting PBMC) wouldnot produce high amounts of empty surface MHC molecules by the coldtemperature procedure.

Harsh acid stripping involves extraction of the peptides withtrifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinitypurified class I-peptide complexes. These methods are not feasible forCTL induction, since it is important to remove the endogenous peptideswhile preserving APC viability and an optimal metabolic state which iscritical for antigen presentation. Mild acid solutions of pH 3 such asglycine or citrate-phosphate buffers have been used to identifyendogenous peptides and to identify tumor associated T cell epitopes.The treatment is especially effective, in that only the MHC class Imolecules are destabilized (and associated peptides released), whileother surface antigens remain intact, including MHC class II molecules.Most importantly, treatment of cells with the mild acid solutions do notaffect the cell's viability or metabolic state. The mild acid treatmentis rapid since the stripping of the endogenous peptides occurs in twominutes at 4° C. and the APC is ready to perform its function after theappropriate peptides are loaded. The technique is utilized herein tomake peptide-specific APCs for the generation of primaryantigen-specific CTL. The resulting APC are efficient in inducingpeptide-specific CD8+CTL.

Activated CD8+ cells may be effectively separated from the stimulatorcells using one of a variety of known methods. For example, monoclonalantibodies specific for the stimulator cells, for the peptides loadedonto the stimulator cells, or for the CD8+ cells (or a segment thereof)may be utilized to bind their appropriate complementary ligand.Antibody-tagged molecules may then be extracted from thestimulator-effector cell admixture via appropriate means, e.g., viawell-known immunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of the activated CD8+ cells can varybetween in vitro and in vivo uses, as well as with the amount and typeof cells that are the ultimate target of these killer cells. The amountwill also vary depending on the condition of the patient and should bedetermined via consideration of all appropriate factors by thepractitioner. Preferably, however, about 1×10⁶ to about 1×10¹², morepreferably about 1×10⁸ to about 1×10¹¹, and even more preferably, about1×10⁹ to about 1×10¹⁰ activated CD8+ cells are utilized for adulthumans, compared to about 5×10⁶-5×10⁷ cells used in mice.

Preferably, as discussed above, the activated CD8+ cells are harvestedfrom the cell culture prior to administration of the CD8+ cells to theindividual being treated. It is important to note, however, that unlikeother present and proposed treatment modalities, the present method usesa cell culture system that is not tumorigenic. Therefore, if completeseparation of stimulator cells and activated CD8+ cells is not achieved,there is no inherent danger known to be associated with theadministration of a small number of stimulator cells, whereasadministration of mammalian tumor-promoting cells may be extremelyhazardous.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8+ cells via intravenous infusion isappropriate.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 A Strategy to Identify Neoepitopes for Vaccination

Our approach to identify tumor-specific neoepitopes involves 3 steps.(1) Identification of DNA mutations using whole genome or whole exome(i.e. only captured exons) sequencing of tumor versus matched germlinesamples from each patient. Our preliminary studies demonstrate that CLLcells contain many distinct genetic changes that alter amino acidsequence and could generate potential novel T cell epitopes. (2)Application of highly validated peptide-MHC binding predictionalgorithms to generate a set of candidate T cell epitopes based onnon-silent mutations present in tumors. We will confirm expression ofmutated genes as RNA in CLL samples, and then confirm the peptide-HLAbinding predictions using an experimental approach to quantify bindingof candidate peptides to HLA alleles. (3) Generation of antigen-specificT cells against mutated peptides.

Example 2 Tumor and Normal Genome Sequencing for the Identification ofMutated Genes in Tumors of Patients with Chronic Lymphocytic Leukemia(Step 1)

To detect tumor-specific mutations (that are not present in normaltissues), samples were collected from tumors and from normal tissues ofeach patient. For leukemias, tumors were purified using magnetic beadisolation or fluorescence-activated cell sorting using antibodiesspecific to tumor cells, e.g., the tumor cells of patients with chroniclymphocytic leukemia (CLL) express the CD5 and CD19 surface markers.Skin fibroblasts were used as a normal tissue control. DNA or RNA forsequencing was purified from isolated tumor or normal tissue cells. Formelanoma, ovarian and other solid tumors (in which there iscontamination with non-tumor cells), DNA and RNA were isolated fromrelatively homogeneous short-term cultures of tumor cells or fromlaser-captured tumor. PBMCs were used as normal control cells. For allsamples, PBMCs were cryopreserved until needed for expansion of mutatedpeptide-specific T cells. Finally, short-term cultures of tumor cellswere also cryopreserved for later use as targets of expanded T cells.Isolated genomic DNA or RNA was tested for nucleic acid integrity andpurity prior to sequencing.

For each sample of DNA, whole genomic DNA was sheared and sequenced, orcoding exons were captured by complementary oligonucleotides usinghybrid selection and then sequenced (Gnirke et al., Nat Biotechnol.2009, 27(2): 182-9). DNA and RNA libraries were generated and sequencedusing Illumina next-generation sequencing instruments.

Sequencing of 64 patients with chronic lymphocytic leukemia (CLL)yielded an average of 23 non-silent mutations that alter protein aminoacid sequences (FIG. 3) in the tumor relative to the germline DNAsequence. These non-silent mutations fall into 5 distinct classes withthe potential to generate neoepitopes: missense, splice-site,frame-shift (indel, insertions and deletions), read-through and genefusions (FIG. 4). The frequencies of these mutations vary acrossindividual patients (FIG. 5). All these mutations provide potentialneoepitopes for immunization, with frame-shift, read-through andsplice-site (e.g. with retained introns) mutations generating longerstretches of novel peptides, missense mutations leading to shortpeptides with single amino acid changes and finally, fusion genesgenerating hybrid peptides with novel junction sequences.

Example 3 Identification of HLA-Binding Peptides Derived from ExpressedProteins Harboring Tumor-Specific Mutations (Step 2)

The next question is whether mutated genes may generate peptides thatcan be presented by patient MHC/HLA proteins. First, several algorithmswere used to predict 30 and 137 HLA-binding peptides with IC50 scores<500 nM from 10 missense mutations of Patient 1, and from 53 missense 1indel and 2 gene fusions of Patient 2. An example for one missensemutation in a patient with 6 specific HLA alleles is shown with 2predicted binding peptides out of 54 combinations of 9-mers peptides andHLA alleles (FIG. 6). To confirm that these genes are expressed intumors, we measure RNA levels for the mutated genes (using severalapproaches that depend on the mutation class, FIG. 7), and found that98% of mutated genes with HLA binding peptides were expressed.

The HLA binding capacity of all predicted peptides that pass RNAexpression validation are then experimentally validated by performingcompetitive binding assays with test peptides versus reference peptidesknown to bind to the HLA allele. (Sidney et al. Curr Protoc Immunol.2001, Chapter 18:Unit 18.3) (FIG. 8A). Of the subset that we submittedfor experimental confirmation of HLA binding, 8 of 17 (47%) predictedpeptides from missense mutations in Pt 1 were confirmed to have highbinding affinities for HLA alleles (IC₅₀<500) (FIG. 8B). For Pt 2, 25 of49 predicted peptides were experimentally confirmed as HLA binding (FIG.8B). These results suggest that all peptides with predicted IC₅₀<150 nMshow HLA binding experimentally, while a cut-off of <500 nM generatestrue binding peptides 40-50% of the time (FIG. 8C). Of note, 12 of the25 confirmed mutated peptides of Pt 2 have >2-fold better bindingaffinity than the germline peptide (FIG. 9). While such peptides arepreferable for incorporating in a tumor vaccine to reduce the chance ofT cells cross-reacting with the germline peptide, peptides that do notshow differential binding may still provide tumor-specific responses dueto differential recognition of mutant vs. germline peptide by the T cellreceptor.

Example 4 CD8+ T Cell Responses Against Mutated Peptides Identified bySequencing CLL Patient Samples (Step 3)

Based on the predicted or experimentally verified HLA-binding mutatedpeptides, we can now determine whether T cells can be generated torecognize these tumor-specific mutated peptides. We thus synthesizedpeptides with binding scores of less than 1000 nM that are derived fromgenes with validated expression in tumor cells. To generate T cells ofdesired specificity, we stimulated T cells of the sequenced patientswith peptide-pulsed (either using an individual peptide or a peptidepool) autologous APCs (dendritic cells and CD40L-expanded autologous Bcells) on a weekly basis, in the presence of IL-2 and IL-7. After 3-4rounds of stimulation, the expanded CD8+ cells were tested on ELISpotfor evidence of reactivity against the peptide, based on IFNgammasecretion. Of the 17 candidate peptides of Patient 1 (FIG. 10), we havedetected IFNgamma secretion in T cells against autologous DCs pulsedwith a mutated peptide from the TLK2 gene.

Example 5 Mutated Bcr-Abl Gene Binds to Patient MHC/HLA Proteins and canElicit Mutant-Peptide-Specific CD8+ T Cells

We performed a more complete study of T cell responses to tumor-specificmutant peptides in patients with another type of leukemia, chronicmyeloid leukemia (CML). CML is defined by the expression of atumor-specific translocation, the product of the BCR-ABL gene fusion.Mutations in BCR-ABL develop in CML patients who develop drug resistanceto front-line pharmacologic therapy with imatinib mesylate, whichtargets BCR-ABL. Potentially, these mutations may generate neoepitopesthat T cells from the host, or an engrafted normal donor, can recognizewhen bound to MHC proteins; these T cells are likely to be minimallytolerized.

We considered the 20 most common mutations that evolve in patients withresistance to imatinib, and predicted the binding of 9- and 10-merpeptides tiled around each mutation. Using either the NetMHC (Nielsen etal. PLoS One. 2007, 2(8):e796) or IEDB (Vita R et al. Nucleic Acids Res.2010, 38:D854-62) predictive algorithms, we predicted binding of 84peptides from 20 common mutations to one or more 8 common HLA alleles(IC₅₀<1000), with many peptides derived from the three most commonmutations. 24 of 84 peptides were predicted to be strong binders(IC₅₀<50) (FIG. 14), 42 peptides intermediate binders (50<IC₅₀<500), and18 peptides weak binders (500<IC₅₀<1000).

We focused our attention on a mutant peptide generated from the E255K(E255K-B₂₅₅₋₂₆₃) mutation (KVYEGVWKK) (SEQ ID NO: 10) that is predictedto bind with high affinity to HLA-A3. (IC₅₀=33.1). Using a competitiveMHC binding assay (FIG. 8A), we experimentally confirmed the highbinding affinity of E255K-B for HLA-A3 (IC₅₀=17 nM) with ˜10-foldstronger HLA-binding of the mutant peptide compared to the parental(wildtype) peptide (FIG. 15A). E255K-B was also experimentally verifiedto bind other A3 supertype family members HLA-A*101 and HLA-A*68. Wenext generated T cell lines against E255K-B from a normal HLA-A3+ donorand 2 E255K+/HLA-A3+CML patients that each demonstrated greaterspecificity against the mutated than the parental peptide (FIG. 15B, C).E255K-B appears to be endogenously processed and presented since T cellsreactive for E255K-B also responded to HLA-A3+APCs transfected with aminigene encompassing 227 base pairs surrounding the E255K mutation.Finally, E255K reactivity in one patient developed only followingcurative allo-HSCT (FIG. 15D). These studies demonstrate thatleukemia-driven genetic alterations can provide novel immunogenictumor-specific antigen targets that are associated with clinicalresponse in vivo. Our approach to identifying immunogenic T cellepitopes of mutated BCR-ABL thus illustrates an effective strategy forapplying bioinformatics tools to discover T cell epitopes from mutatedgenes.

Example 6 Patient T Cell Clones that Recognize Tumor Epitopes canSelectively Kill Cells Presenting Mutated Epitopes

Confirmation of target specificity of T cells is best addressed bycharacterization of individual T cell clones. We therefore typicallyisolate mutated peptide-specific T cell clones by limiting dilution ofreactive T cell lines and then use standard chromium release assays toscreen for T cell clones that demonstrate differential killing ofmutated vs germ line peptide-pulsed autologous APCs. Using a standarddilution series for each peptide, we measure the concentration ofpeptide required for 50% killing. If the ratio of wild type to mutantpeptides needed for 50% killing is greater than 10-fold, we concludethat there is differential recognition of these peptides by T cells, asseen previously for mutated tumor antigens. We have carried out thisprocedure for a CML tumor antigen, CML66. To determine whetherCML66-peptide-specific T cells recognize processed and presentedepitopes, CML66-peptide-reactive T cells were incubated with autologousAPCs transduces to express the entire CML66 proetin. We expressed CML66by nucleofection of either plasmid DNA, or in vitro transcribed RNA (inDCs, CD40L-expanded B cells, or K562 cells with engineered HLAmolecules). As shown in FIG. 12A, stimulated T cells were specific toHLA-B4403 bound CML66-derived peptide epitope (peptide 66-72C). Sincewhole CML66 protein was efficiently expressed when CD40L-expanded Bcells were nucleofected with CML66 mRNA (FIG. 12B), we were able to usethese cells (or peptide pulsed cells) as targets in a standard chromiumrelease assay and found that the T cells lysed these targets celleffectively (FIG. 12C). Comparable assays, including lysing ofpatient-matched tumor cells, are being carried out for each of themutated peptide-specific T cell lines generated from each cancer patient(e.g. using the T cell lines described in Examples 6 and 7).

Example 7 Mutated Tumor Drivers as Potential Tumor Antigens

Of 1188 nonsilent mutations across 64 patients, we identified 8recurrent mutations, including SF3B1 (16% of CLL patients), TP53(12.5%), MYD88 (9%), ATM (9%), FBXW7 (6%), MAPK1 (5%), GNB1 (3%) andM6PR (3%) (FIG. 11). These mutations (especially the most frequent ones:SF3B1, TP53, MYD88 and ATM) are predicted to be driver mutations thatare essential for tumor development or progression. These driver genesrepresent promising tumor-specific antigens for inclusion in a vaccine.

SF3B1 is the most frequently mutated gene in CLL, is mutated atconserved sites, is highly expressed in CLL patients (FIG. 12), and hasnot been previously described. The most common SF3B1 mutation was K700E(40% of SF3B1 mutations); genotyping of an additional 89 independent CLLpatients uncovered 6 more patient tumors harboring this mutation. Byapplying peptide-HLA binding algorithms to the SF3B1 mutations, wepredict binding of the mutated peptides to the most common HLA-A2 allele(FIG. 13). If a peptide that harbors the most common mutation in CLL(SF3B1 K700E) binds the most common class I HLA allele (HLA-A2), thenthis peptide is an excellent candidate for inclusion in a CLL vaccinefor many CLL patients.

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We claim:
 1. A method of identifying a neoantigen comprising: a.identifying a tumor specific mutation in an expressed gene of a subjecthaving cancer, b. wherein when said mutation identified in step (a) is apoint mutation: i. identifying a mutant peptide having the mutationidentified in step (a), wherein said mutant peptide binds to a class IHLA protein with a greater affinity than a wild-type peptide; and has anIC50 less than 500 nm; c. wherein when said mutation identified in step(a) is a splice-site, frameshift, read-through or gene-fusion mutation:i. identifying a mutant polypeptide encoded by the mutation identifiedin step (a), wherein said mutant polypeptide binds to a class I HLAprotein.
 2. The method of claim 1, wherein the mutant peptide is about8-10 amino acids in length.
 3. The method of claim 1, wherein the mutantpeptide is greater than 10 amino acids in length.
 4. The method of claim3, wherein the mutant peptide is greater than 15 amino acids in length.5. The method of claim 4, wherein the mutant peptides is greater than 20amino acids in length.
 6. The method of claim 5, wherein the mutantpeptides is greater than 30 amino acids in length.
 7. The method ofclaim 1, wherein the mutant peptides is about 8 to 50 amino acids inlength.
 8. The method of claim 1, wherein the mutant peptides is about24-40 amino acids in length.
 9. The method of claim 1, wherein tumorspecific mutations are identified by nucleic acid sequencing.
 10. Themethod of claim 1, further comprising selecting a peptide identified instep (b) or the polypeptide of step (c) that activates anti-tumor CD8 Tcells.
 11. A method of inducing a tumor specific immune response in asubject comprising administering one or more peptides or polypeptidesidentified according to claim 1 and an adjuvant.
 12. The method of claim11, wherein the adjuvant is a TLR-based adjuvant.
 13. The method ofclaim 11, wherein the peptide or polypeptide is emulsified with amineral-oil based adjuvant.
 14. The method of claim 11, wherein thepeptide or polypeptide and a TLR-based adjuvant are emulsified with amineral-oil based adjuvant.
 15. The method of claim 11, furthercomprising administering an anti-immunosuppressive agent.
 16. The methodof claim 15, wherein the anti-immunosuppressive agent is an anti-CTLA-4antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-CD25antibody or an inhibitor of IDO.
 17. A method of inducing a tumorspecific immune response in a subject comprising administering to thesubject autologous dendritic cells or antigen presenting cells that havebeen pulsed with one or more of the peptides or polypeptides identifiedaccording to claim
 1. 18. The method of claim 17, further comprisingadministering an adjuvant.
 19. The method of claim 18, wherein theadjuvant is a TLR-based adjuvant.
 20. The method of claim 17, furthercomprising administering an anti-immunosuppressive agent.
 21. The methodof claim 20, wherein said anti-immunosuppressive agent is an anti-CTLA-4antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody, an anti-CD25antibody or an inhibitor of IDO.
 22. A method of vaccinating or treatinga subject for cancer comprising: a. identifying a plurality of tumorspecific mutations in an expressed gene of the subject wherein when saidmutation identified is a: i. point mutation further identifying a mutantpeptide having the point mutation; and/or ii. splice-site, frameshift,read-through or gene-fusion mutation further identifying a mutantpolypeptide encoded by the mutation; b. selecting one or more mutantpeptides or polypeptides identified in step (a) that binds to a class IHLA protein; c. selecting the one or more mutant peptides orpolypeptides identified in step (b) that is capable of activatinganti-tumor CD8 T-cells; and d. administering to the subject the one ormore peptides or polypeptides, autologous dendritic cells or antigenpresenting cells pulsed with the one or more peptides or polypeptidesselected in step (c).
 23. The method of claim 22, further comprisingadministering the subject an adjuvant.
 24. The method of claim 23,wherein the adjuvant is a TLR-based adjuvant.
 25. The method of claim22, further comprising administering an anti-immunosuppressive agent.26. The method of claim 25, wherein said anti-immunosuppressive agent isan anti-CTLA-4 antibody, an anti-PD 1 antibody, an anti-PD-L1 antibody,an anti-CD25 antibody or an inhibitor of IDO.
 27. The method of claim22, wherein the mutant peptide is about 8-10 amino acids in length. 28.The method of claim 22, wherein the mutant peptides is about 8 to 50amino acids in length.
 29. The method of claim 22, wherein the mutantpeptide is about 24-40 amino acids in length.
 30. The method of claim22, wherein said subject is has received a hematopoictic stem celltransplant.
 31. The method of claim 22, wherein the subject is a human,dog, cat, or horse.
 32. The method of claim 22, wherein the cancer isbreast cancer, ovarian cancer, prostate cancer, lung cancer, kidneycancer, gastric cancer, colon cancer, testicular cancer, head and neckcancer, pancreatic cancer, brain cancer, melanoma lymphoma or leukemia.33. The method of claim 32, wherein the lymphoma is a B cell lymphoma.34. The method of claim 32, wherein the leukemia is acute myelogenousleukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, orT cell lymphocytic leukemia.
 35. A pharmaceutical composition comprisingthe peptide identified according claim 1 and a pharmaceuticallyacceptable carrier.
 36. A composition comprising at least two distinct:a. SF3B1 peptides wherein each peptide is equal to or less than 50 aminoacids in length and contains i. a leucine at amino acid position 625;ii. a histidine at amino acid position 626; iii. a glutamic acid atamino acid position 700; iv. an aspartic acid at amino acid position742; or v. an arginine at amino acid position 903, when numbered inaccordance with wild-type SF3B1; b. MYD88 peptides wherein each peptideis equal to or less than 50 amino acids in length and contains i. athreonine at amino acid position 232; ii. a leucine at amino acidposition 258; or iii. a proline at amino acid position 265, whennumbered in accordance with wild-type MYD88; c. TP53 peptides whereineach peptide is equal to or less than 50 amino acids in length andcontains i. an arginine at amino acid position 111; ii. an arginine atamino acid position 215; iii. a serine at amino acid position 238; iv. aglutamine at amino acid position 248; v. a phenylalanine at amino acidposition 255; vi. a cysteine at amino acid position 273 or vii. anasparagine at amino acid position 281, when numbered in accordance withwild-type TP53; d. ATM peptides wherein each peptide is equal to or lessthan 50 amino acids in length and contains i. a phenylalanine at aminoacid position 1252; ii. an arginine at amino acid position 2038; iii. ahistidine at amino acid position 2522; or iv. a cysteine at amino acidposition 2954, when numbered in accordance with wild-type ATM; e. ablpeptides wherein each peptide is equal to or less than 50 amino acids inlength and contains i. a valine at amino acid position 244; ii. a valineat amino acid position 248; iii. a glutamic acid at amino acid position250; iv. an alanine at amino acid position 250; v. a histidine at aminoacid position 252; vi. an arginine at amino acid position 252; vii. aphenylalanine at amino acid position 253; viii. a histidine at aminoacid position 253; ix. a lysine at amino acid position 255; x. a valineat amino acid position 255; xi. a glycine at amino acid position 276;xii. an isoleucine at amino acid position 315; xiii. an asparagine atamino acid position 315; xiv. a leucine at amino acid position 317; xv.a threonine at amino acid position 343; xvi. a threonine at amino acidposition 351; xvii. a glycine at amino acid position 355; xviii. avaline at amino acid position 359; xix. an alanine at amino acidposition 359; xx. an isoleucine at amino acid position 379; xxi. aleucine at amino acid position 382; xxii. a methionine at amino acidposition 387; xxiii. a proline at amino acid position 396; xxiv. anarginine at amino acid position 396; xxv. a tyrosine at amino acidposition 417; or xxvi. a serine at amino acid position 486, whennumbered in accordance with wild-type abl; f. FBXW7 peptides whereineach peptide is equal to or less than 50 amino acids in length andcontains: i. a leucine at amino acid position 280; ii. a histidine atamino acid position 465; iii. a cysteine at amino acid position 505; oriv. a glutamic acid at amino acid position 597, when numbered inaccordance with wild-type FBXW7; g. MAPK1 peptides wherein each peptideis equal to or less than 50 amino acids in length and contains i. anasparagine at amino acid position 162; ii. a glycine at amino acidposition 291; or iii. a phenylalanine at amino acid position 316, whennumbered in accordance with wild-type MAPK1; or h. GNB1 peptides whereineach peptide is equal to or less than 50 amino acids in length andcontains a threonine at amino acid position 180, when numbered inaccordance with wild-type GNB1.
 37. The composition of claim 36, furthercomprising an adjuvant.
 38. A method of treating a subject with animatinib resistant tumor comprising administering to a HLA-A3 positivesubject a composition a Bcr-abl peptide equal to or less than 50 aminoacid in length that contains a lysine at position 255 when numbered inaccordance with wild-type bcr-abl.
 39. A method of treating a subjectwith an imatinib resistant tumor comprising administering to saidsubject one or more peptides containing a bcr-abl mutation wherein saidpeptide is equal to or less than 50 amino acid and binds to a class IHLA protein with an IC50 less than 500 nm.